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EP3808778A1 - Procédés d'augmentation des taux de globules rouges et de traitement de la drépanocytose - Google Patents

Procédés d'augmentation des taux de globules rouges et de traitement de la drépanocytose Download PDF

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EP3808778A1
EP3808778A1 EP20197941.6A EP20197941A EP3808778A1 EP 3808778 A1 EP3808778 A1 EP 3808778A1 EP 20197941 A EP20197941 A EP 20197941A EP 3808778 A1 EP3808778 A1 EP 3808778A1
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polypeptide
amino acid
subject
seq
activin
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Ravindra Kumar
Naga Venkata Sai Rajasekhar SURAGANI
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Acceleron Pharma Inc
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Acceleron Pharma Inc
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • A61K31/16—Amides, e.g. hydroxamic acids
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    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4412—Non condensed pyridines; Hydrogenated derivatives thereof having oxo groups directly attached to the heterocyclic ring
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    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61P15/10—Drugs for genital or sexual disorders; Contraceptives for impotence
    • A—HUMAN NECESSITIES
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    • A61P27/00—Drugs for disorders of the senses
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A—HUMAN NECESSITIES
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    • A61P7/06—Antianaemics
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
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    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • Hematopoiesis is the formation of cellular components of the blood from self-renewing hematopoietic stem cells located mainly in the bone marrow, spleen, or lymph nodes during postnatal life. Blood cells can be classified as belonging to the lymphocytic lineage, myelocytic lineage, or erythroid lineage. By a process known as lymphopoiesis, common lymphoid progenitor cells give rise to T-cells, B-cells, natural killer cells, and dendritic cells.
  • myelopoiesis By a process termed myelopoiesis, common myeloid progenitor cells give rise to macrophages, granulocytes (basophils, neutrophils, eosinophils, and mast cells), and thrombocytes (platelets). Finally, by a process known as erythropoiesis, erythroid progenitor cells give rise to red blood cells (erythrocytes).
  • Postnatal erythropoiesis occurs primarily in the bone marrow and in the red pulp of the spleen.
  • the coordinated action of various signaling pathways controls the balance of cell proliferation, differentiation, survival, and death.
  • red blood cells are produced at a rate that maintains a constant red cell mass in the body, and production may increase or decrease in response to various stimuli, including increased or decreased oxygen tension or tissue demand.
  • the process of erythropoiesis begins with the formation of lineage-committed precursor cells and proceeds through a series of distinct precursor cell types. The final stages of erythropoiesis occur as reticulocytes are released into the bloodstream and lose their mitochondria and ribosomes while assuming the morphology of mature red blood cell.
  • red blood cell production rates An elevated level of reticulocytes, or an elevated reticulocyte:erythrocyte ratio, in the blood is indicative of increased red blood cell production rates.
  • the mature red blood cell (RBC) is responsible for oxygen transport in the circulatory systems of vertebrates. Red blood cells contain high concentrations of hemoglobin, a protein that binds to oxygen in the lungs at relatively high partial pressure of oxygen (pO 2 ) and delivers oxygen to areas of the body with relatively low pO 2 .
  • Erythropoietin is widely recognized as a significant positive regulator of postnatal erythropoiesis in vertebrates. EPO regulates the compensatory erythropoietic response to reduced tissue oxygen tension (hypoxia) and low red blood cell levels or low hemoglobin levels. In humans, elevated EPO levels promote red blood cell formation by stimulating the generation of erythroid progenitors in the bone marrow and spleen. In the mouse, EPO enhances erythropoiesis primarily in the spleen.
  • EPO Effects of EPO are mediated by a cell-surface receptor belonging to the cytokine receptor superfamily.
  • the human EPO receptor gene encodes a 483 amino acid transmembrane protein; however, the active EPO receptor is thought to exist as a multimeric complex even in the absence of ligand (see, e.g., U.S. Pat. No. 6,319,499 ).
  • the cloned full-length EPO receptor expressed in mammalian cells binds EPO with an affinity similar to that of the native receptor on erythroid progenitor cells.
  • Binding of EPO to its receptor causes a conformational change resulting in receptor activation and biological effects including increased proliferation of immature erythroblasts, increased differentiation of immature erythroblasts, and decreased apoptosis in erythroid progenitor cells [see, e.g., Liboi et al. (1993) Proc Natl Acad Sci USA 90:11351-11355 ; Koury et al. (1990) Science 248:378-381 ].
  • Anemia is a broadly-defined condition characterized by lower than normal levels of hemoglobin or red blood cells in the blood.
  • anemia is caused by a primary disorder in the production or survival of red blood cells (e.g., sickle-cell anemia). More commonly, anemia is secondary to diseases of other systems [see, e.g ., Weatherall & Provan (2000) Lancet 355, 1169-1175 ].
  • Anemia may result from a reduced rate of production or increased rate of destruction of red blood cells or by loss of red blood cells due to bleeding.
  • Anemia may result from a variety of disorders that include, for example, acute or chronic renal failure or end stage renal disease, chemotherapy treatment, a myelodysplastic syndrome, rheumatoid arthritis, and bone marrow transplantation.
  • EPO Errorless blood cell
  • Treatment with EPO typically causes a rise in hemoglobin by about 1-3 g/dL in healthy humans over a period of weeks. When administered to anemic individuals, this treatment regimen often provides substantial increases in hemoglobin and red blood cell levels and leads to improvements in quality of life and prolonged survival.
  • EPO is not uniformly effective, and many individuals are refractory to even high doses [see, e.g., Horl et al. (2000) Nephrol Dial Transplant 15, 43-50 ].
  • EPO is not uniformly effective, and many individuals are refractory to even high doses [see, e.g., Horl et al. (2000) Nephrol Dial Transplant 15, 43-50 ].
  • end-stage renal disease are hyporesponsive to EPO [see, e.g., Glaspy et al. (1997) J Clin Oncol 15, 1218-1234 ; Demetri et al.
  • EPO-based therapeutic compounds e.g ., erythropoietin-stimulating agents, ESAs
  • ESAs erythropoietin-stimulating agents
  • Sickle-cell disease is a hereditary blood disorder characterized by red blood cells that assume an abnormal, rigid, sickle shape [see, e.g., Eaton et al. (1990) Adv Protein Chem, 40: 63-279 ; Steinberg, MH (1999) N Engl J Med 340(13): 1021-1030 ; and Ballas et al. (1992) Blood, 79(8) 2154-63 ].
  • the loss of red blood cell elasticity is believed to be central to the pathophysiology of sickle-cell disease. Normal red blood cells are quite elastic, which allows the red blood cells to deform while passing through capillaries.
  • sickle-cell disease In sickle-cell disease, low-oxygen tension promotes red blood cell sickling, and repeated episodes of sickling damage the cell membrane and thus decreases the cell's elasticity. Furthermore, sickle-cells often fail to return to a normal shape when normal oxygen tension is restored. As a consequence, these rigid blood cells are unable to deform as they pass through narrow capillaries, resulting in vascular (vaso) occlusion and ischemia. Furthermore, sickle-cells are more prone to hemolysis than normal red blood cells, resulting in a high incidence of anemia in subjects with sickle-cell disease.
  • Sickle-cell disease is characterized by various acute and chronic complications, which are associated with significant morbidity and mortality in afflicted subject [see, e.g., Kassim et al. (2013) Annu Rev Med, 64: 451-466 ].
  • the terms "sickle-cell crisis” or “sickling crisis” may be used to describe several independent acute complications occurring in patients with sickle-cell disease including, for example, pain crisis, painful crisis, anemia crisis, vaso-occlusive crisis, aplastic crisis, sequestration crisis ( e.g ., splenic and/or liver), and hemolytic crisis.
  • EPO-based therapeutics have been observed to aggravate other aspects of sickle-cell disease, such as vaso-occlusive crisis (e.g ., leading to an increase in vaso-occlusive pain and/or hypertension) [see, e.g., Little et al. (2006) Haematologica, 91(8): 1076-1083 ].
  • the disclosure provides methods of treating sickle-cell disease (SCD), particularly treating or preventing one or more complications of SCD, with one or more ActRII antagonists.
  • ActRII antagonists of the disclosure include, for example, agents that can inhibit ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor) mediated activation of a signal transduction pathway (e.g ., activation of signal transduction via intracellular mediators, such as SMAD 1, 2, 3, 5, and/or 8); agents that can inhibit one or more ActRII ligands (e.g ., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.
  • ActRII antagonists of the disclosure include, for example, agents that can inhibit ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor) mediated activation of a signal transduction pathway (e.g ., activation of signal transduction
  • agents that inhibit expression e.g ., transcription, translation, cellular secretion, or combinations thereof
  • agents that can inhibit one or more intracellular mediators of the ActRII signaling pathway e.g ., SMADs 1, 2, 3, 5, and/or 8.
  • the disclosure concerns an ActRII antagonist for use in a method for treating sickle-cell disease in a subject in need thereof.
  • the disclosure concerns an ActRII antagonist for the manufacture of a medicament for treating sickle-cell disease in a subject in need thereof.
  • the disclosure concerns an ActRII antagonist for use in a method for treating one or more complications of sickle-cell disease.
  • the disclosure concerns an ActRII antagonist for the manufacture of a medicament for treating one or more complications sickle-cell disease in a subject in need thereof.
  • the disclosure concerns an ActRII antagonist for use in a method for preventing one or more complications of sickle-cell disease.
  • the disclosure concerns an ActRII antagonist for the manufacture of a medicament for preventing one or more complications sickle-cell disease in a subject in need thereof.
  • the disclosure provides methods for using an ActRII antagonist, or combination of ActRII antagonists, to treat or prevent one or more complications of sickle-cell disease including, for example, anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload and complications of iron overload ( e.g ., congestive heart failure, cardiac arrhythmia, myocardial infarction, other forms of cardiac disease, diabetes mellitus, dyspnea, hepatic disease and adverse effects of iron chelation therapy), splenic infarction, acute and/or chronic renal failure, pyelonephritis
  • the disclosure provides methods for using an ActRII antagonist, or combination of ActRII antagonists, to treat or prevent vascular occlusion (vaso-occlusion) in a sickle-cell disease patient in need thereof as well as various complications associated with vaso-occlusion in a sickle-cell disease patient ( e.g., vaso-occlusion crisis, pain crisis, etc. ) .
  • the disclosure provides methods for using an ActRII antagonist, or combination of ActRII antagonists, to treat or prevent anemia in a sickle-cell disease patient in need thereof as well as various complications associated with anemia in a sickle-cell disease patient ( e.g., aplastic crisis, hyperhemolytic crisis, etc. ) .
  • ActRII antagonists can be used to increase red blood cell levels while reducing the need for red blood cell transfusions and/or iron chelation therapy, and thereby reduce morbidity and mortality associated with iron accumulation in vulnerable tissues/organs.
  • ActRII antagonists can also be used to reduce the need for other supportive therapies for treating sickle-cell disease [ e.g ., treatment with hydroxyurea, treatment with an EPO or other EPO agonist, and/or pain management (e.g ., treatment with one or more of opioid analgesic agents, non-steroidal anti-inflammatory drugs, and/or corticosteroids)].
  • ActRII antagonists can be used in combination with existing supportive therapies for sickle-cell disease including, for example, transfusion of red blood cells, iron chelation therapy, hydroxyurea therapy, EPO or EPO agonist therapy, and/or pain management therapy.
  • ActRII antagonists of the disclosure can be used to reduce the amount, duration, etc. of an existing supportive therapy for sickle-cell disease.
  • transfusion of red blood cells and iron chelation therapy may help treat certain complications of sickle-cell disease, they sometimes result in adverse side effects. Therefore, in certain aspects, ActRII antagonists as described herein can be used to reduce amount of a second supportive therapy, e.g ., reduce blood cell transfusion burden or reduce the dosage of a chelation therapeutic.
  • the disclosure provides uses of an ActRII antagonist, or combination of ActRII antagonists, (optionally in combination with one or more supportive therapies for sickle-cell disease) for making a medicament for the treatment or prevention of sickle-cell disease, particularly one or more complications of sickle-cell disease as disclosed herein.
  • the examples of the instant application demonstrate that ActRII antagonists can be used to increase various red blood cell parameters (e.g ., red blood cell levels, hemoglobin levels, hematocrit levels, etc. ) and treat anemia resulting from various disorders/conditions.
  • the examples of the disclosure further show that an ActRII antagonist can be used to treat sickle-cell disease.
  • a GDF trap which comprises a soluble, extracellular domain of an ActRIIB polypeptide having an acidic amino acid at position 79 with respect to SEQ ID NO:1
  • the data herein indicates that ActRII antagonists can be used to treat or prevent anemia in sickle-cell disease patients.
  • the GDF trap also improves red blood cell morphology.
  • This observed improvement in red blood cell morphology indicates that ActRII antagonist therapy also may be used to treat or prevent various non-anemia complications of sickle-cell disease including, for example, complications arising from vascular occlusion (also referred to as vaso-occlusion). In some instances, these associated complications are of equal or greater importance to health and quality of life in patients with sickle-cell disease than the anemic condition.
  • ActRII antagonists of the present disclosure can be used to treat or prevent various complications of sickle-cell disease including, for example, anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage, intraventricular hemorrhage, peripheral retinal ischemia, proliferative sickle retinopathy, vitreous hemorrhage, and/or
  • preferred ActRII antagonists to be used in accordance with the methods disclosed herein are agents that bind to and/or inhibit GDF11 and/or GDF8 (e.g., an agent that inhibits GDF11- and/or GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling). Such agents are referred to collectively as GDF-ActRII antagonists.
  • GDF-ActRII antagonists may further inhibit one or more of activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, and Nodal.
  • the disclosure provides methods of using one or more ActRII antagonists, including, for example, soluble ActRIIA polypeptides, soluble ActRIIB polypeptides, GDF trap polypeptides, anti-ActRIIA antibodies, anti-ActRIIB antibodies, anti-ActRII ligand antibodies (e.g , anti-GDF1 antibodies, anti-GDF8 antibodies, anti-activin A antibodies, anti-activin B antibodies, anti-activin AB antibodies, anti-activin C antibodies, anti-activin E antibodies, anti-BMP6 antibodies, anti-BMP7 antibodies, and anti-Nodal antibodies), small-molecule inhibitors of ActRIIA, small-molecule inhibitors of ActRIIB, small-molecule inhibitors of one or more ActRII ligands (e.g ., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.
  • ActRII antagonists including, for example, soluble Act
  • ActRII ligands e.g ., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, Nodal, etc.
  • ActRII antagonists to be used in accordance with the methods disclosed herein do not substantially bind to and/or inhibit activin A (e.g ., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • activin A e.g ., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • an ActRII antagonist comprising a variant, extracellular (soluble) ActRIIB domain that binds to and inhibits GDF11 activity (e.g., GDF11-mediated ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling) may be used to increase red blood cell levels in vivo, treat anemia resulting from various conditions/disorders, and treat sickle-cell disease (e.g ., increase red blood cell levels and improve red blood cell morphology in sickle-cell disease patients).
  • GDF11 activity e.g., GDF11-mediated ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling
  • preferred ActRII antagonists to be used in accordance with the methods disclosed herein are soluble ActRII polypeptides (e.g. soluble ActRIIA or ActRIIB polypeptides) that bind to and/or inhibit GDF11 (e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • soluble ActRII polypeptides e.g. soluble ActRIIA or ActRIIB polypeptides
  • GDF11 e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • soluble ActRIIA and soluble ActRIIB ActRII antagonists may affect red blood cell formation and/or morphology through a mechanism other than GDF11 antagonism, the disclosure nonetheless demonstrates that desirable therapeutic agents, with respect to the methods disclosed herein, may be selected on the basis of GDF11 antagonism or ActRII antagonism or both.
  • such soluble ActRII polypeptide antagonist may further bind to and/or inhibit GDF8 (e.g . inhibit GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • soluble ActRIIA and ActRIIB polypeptides of the disclosure that bind to and/or inhibit GDF11 and/or GDF8 may further bind to and/or inhibit one or more additional ActRII ligands selected from: activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal.
  • the present disclosure provides GDF traps that are variant ActRII polypeptides (e.g ., ActRIIA and ActRIIB polypeptides), including ActRII polypeptides having amino- and carboxy-terminal truncations and/or other sequence alterations (one or more amino acid substitutions, additions, deletions, or combinations thereof).
  • GDF traps of the invention may be designed to preferentially antagonize one or more ligands of ActRII receptors, such as GDF8 (also called myostatin), GDF11, Nodal, BMP6, and BMP7 (also called OP-1).
  • examples of GDF traps include a set of variants derived from ActRIIB that have greatly diminished affinity for activin, particularly activin A. These variants exhibit desirable effects on red blood cells while reducing effects on other tissues. Examples of such variants include those having an acidic amino acid [ e.g., aspartic acid (D) or glutamic acid (E)] at the position corresponding to position 79 of SEQ ID NO:1.
  • an acidic amino acid e.g., aspartic acid (D) or glutamic acid (E)
  • preferred GDF traps to be used in accordance with the methods disclosed herein bind to and/or inhibit GDF11.
  • GDF11 binds to and/or inhibit GDF8.
  • GDF traps that bind to and/or inhibit GDF11 and/or GDF8 may further bind to and/or inhibit one or more additional ActRII ligands (e.g ., activin B, activin E, BMP6, BMP7, and Nodal).
  • additional ActRII ligands e.g ., activin B, activin E, BMP6, BMP7, and Nodal.
  • GDF traps to be used in accordance with the methods disclosed herein to not substantially bind to and/or inhibit activin A e.g ., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • a GDF trap polypeptide comprises an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NOs: 36, 37, 41, 44, 45, 50 or 51, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing.
  • a GDF trap polypeptide comprises an amino acid sequence that comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 10, 11, 22, 26, 28, 29, 31, or 49, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing.
  • a GDF trap polypeptide comprises an amino acid sequence that comprises of the amino acid sequence of SEQ ID NOs: 2, 3, 4, 5, 6, 29, 31, or 49, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing, wherein the position corresponding to 79 in SEQ ID NO: 1, 4, or 50 is an acidic amino acid.
  • a GDF trap may include a functional fragment of a natural ActRII polypeptide, such as one comprising at least 10, 20, or 30 amino acids of a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 9, 10, 11, or 49 or a sequence of SEQ ID NO: 2, 5, 10, 11, or 49 lacking the C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at the N-terminus.
  • a preferred polypeptide will comprise a truncation relative to SEQ ID NO: 2 or 5 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus.
  • a preferred GDF trap for use in such a preparation consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 36.
  • a GDF trap comprising an altered ActRII ligand-binding domain has a ratio of K d for activin A binding to K d for GDF11 and/or GDF8 binding that is at least 2-, 5-, 10-, 20, 50-, 100-, or even 1000-fold greater relative to the ratio for the wild-type ligand-binding domain.
  • the GDF trap comprising an altered ligand-binding domain has a ratio of IC 50 for inhibiting activin A to IC 50 for inhibiting GDF11 and/or GDF8 that is at least 2-, 5-, 10-, 20-, 25- 50-, 100-, or even 1000-fold greater relative to the wild-type ActRII ligand-binding domain.
  • the GDF trap comprising an altered ligand-binding domain inhibits GDF11 and/or GDF8 with an IC 50 at least 2, 5, 10, 20, 50, or even 100 times less than the IC 50 for inhibiting activin A.
  • These GDF traps can be fusion proteins that include an immunoglobulin Fc domain (either wild-type or mutant).
  • the subject soluble GDF traps are antagonists (inhibitors) of GDF8 and/or GDF11.
  • the disclosure provides GDF traps which are soluble ActRIIB polypeptides comprising an altered ligand-binding (e.g ., GDF11-binding) domain.
  • GDF traps with altered ligand-binding domains may comprise, for example, one or more mutations at amino acid residues such as E37, E39, R40, K55, R56, Y60, A64, K74, W78, L79, D80, F82 and F101 of human ActRIIB (numbering is relative to SEQ ID NO: 1).
  • the altered ligand-binding domain can have increased selectivity for a ligand such as GDF8/GDF11 relative to a wild-type ligand-binding domain of an ActRIIB receptor.
  • a ligand such as GDF8/GDF11
  • GDF8/GDF11 a wild-type ligand-binding domain of an ActRIIB receptor.
  • these mutations are demonstrated herein to increase the selectivity of the altered ligand-binding domain for GDF11 (and therefore, presumably, GDF8) over activin: K74Y, K74F, K74I, L79D, L79E, and D80I.
  • the following mutations have the reverse effect, increasing the ratio of activin binding over GDF11: D54A, K55A, L79A and F82A.
  • the overall (GDF11 and activin) binding activity can be increased by inclusion of the "tail" region or, presumably, an unstructured linker region, and also by use of a K74A mutation.
  • Other mutations that caused an overall decrease in ligand binding affinity include: R40A, E37A, R56A, W78A, D80K, D80R, D80A, D80G, D80F, D80M and D80N. Mutations may be combined to achieve desired effects. For example, many of the mutations that affect the ratio of GDF11: activin binding have an overall negative effect on ligand binding, and therefore, these may be combined with mutations that generally increase ligand binding to produce an improved binding protein with ligand selectivity.
  • a GDF trap is an ActRIIB polypeptide comprising an L79D or L79E mutation, optionally in combination with additional amino acid substitutions, additions, or deletions.
  • ActRII antagonists to be used in accordance with the methods disclosed herein are ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides.
  • ActRIIB polypeptides and ActRIIB-based GDF trap polypeptides are soluble polypeptides that comprise a portion/domain derived from the ActRIIB sequence of SEQ ID NO:1, 4, or 49, particularly an extracellular, ligand-binding portion/domain derived from the ActRIIB sequence of SEQ ID NO:1, 4, or 49.
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 21-29 ( e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO:1 or 4 [optionally beginning at 22-25 ( e.g., 22, 23, 24, or 25) of SEQ ID NO:1 or 4] and ending at any one of amino acids 109-134 ( e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4.
  • amino acids 21-29 e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29
  • 109-134 e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-29 ( e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 [optionally beginning at 22-25 ( e.g., 22, 23, 24, or 25) of SEQ ID NO:1 or 4] and ending at any one of amino acids 109-133 ( e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4.
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-24 ( e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 [optionally beginning at 22-25 ( e.g., 22, 23, 24, or 25) of SEQ ID NO:1 or 4] and ending at any one of amino acids 109-133 ( e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4.
  • amino acids 20-24 e.g., 20, 21, 22, 23, or 24
  • 22-25 e.g., 22, 23, 24, or 25
  • amino acids 109-133 e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 21-24 ( e.g., 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any of amino acids 109-134 (e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4.
  • amino acids 21-24 e.g., 21, 22, 23, or 24
  • amino acids 109-134 e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-24 ( e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 118-133 ( e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 21-24 ( e.g., 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 118-134 ( e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4.
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-24 ( e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 128-133 ( e.g., 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4.
  • the portion derived from ActRIIB corresponds to a sequence beginning at any of amino acids 20-24 ( e.g., 20, 21, 22, 23, or 24) of SEQ ID NO: 1 or 39 and ending at any of amino acids 128-133 ( e.g., 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 39.
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 21-29 ( e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 118-134 ( e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4.
  • amino acids 21-29 e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29
  • amino acids 118-134 e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-29 ( e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 118-133 ( e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4.
  • amino acids 20-29 e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29
  • amino acids 118-133 e.g., 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133
  • the portion derived from ActRIIB corresponds to a sequence beginning at one any of amino acids 21-29 ( e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 128-134 ( e.g., 128, 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1 or 4.
  • amino acids 21-29 e.g., 21, 22, 23, 24, 25, 26, 27, 28, or 29
  • amino acids 128-134 e.g., 128, 129, 130, 131, 132, 133, or 134
  • the portion derived from ActRIIB corresponds to a sequence beginning at any one of amino acids 20-29 ( e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 or 4 and ending at any one of amino acids 128-133 ( e.g., 128, 129, 130, 131, 132, or 133) of SEQ ID NO: 1 or 4.
  • ActRIIB and ActRIIB-based GDF trap constructs beginning at 22-25 ( e.g., 22, 23, 24, or 25) of SEQ ID NO: 1 or 4 have activity levels greater than proteins having the full extracellular domain of human ActRIIB.
  • the ActRIIB polypeptides and ActRIIB-based GDF trap polypeptides comprise, consist essentially of, or consist of, an amino acid sequence beginning at amino acid position 25 of SEQ ID NO: 1 or 4 and ending at amino acid position 131 of SEQ ID NO: 1 or 4.
  • Any of the foregoing ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides may be produced as a homodimer.
  • Any of the foregoing ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides may further comprise a heterologous portion that comprises a constant region from an IgG heavy chain, such as an Fc domain.
  • any of the above ActRIIB-based GDF trap polypeptides may comprise an acidic amino acid at the position corresponding to position 79 of SEQ ID NO: 1, optionally in combination with one or more additional amino acid substitutions, deletions, or insertions relative to SEQ ID NO: 1.
  • Any of the above ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by activin (e.g ., activin A, activin B, activin C, or activin AB) in a cell-based assay.
  • activin e.g ., activin A, activin B, activin C, or activin AB
  • any of the above ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by GDF11 and/or GDF8 in a cell based assay.
  • any of the above ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides, including homodimer and/or fusion proteins thereof may bind to and/or inhibit signaling of one or more of activin B, activin C, activin E, BMP6, BMP7, and Nodal in a cell-based assay.
  • ActRIIB polypeptides and ActRIIB-based GDF Trap polypeptides are contemplated, such as the following.
  • An ActRIIB polypeptide or GDF trap polypeptide comprising an amino acid sequence that is at least 80% ( e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 4, wherein the position corresponding to 64 of SEQ ID NO: 1 is an R or K, and wherein the ActRIIB polypeptide or ActRIIB-based GDF trap polypeptide inhibits signaling by activin, GDF8, and/or GDF11 in a cell-based assay.
  • ActRIIB polypeptide or ActRIIB-based GDF trap polypeptide as above wherein at least one alteration with respect to the sequence of SEQ ID NO: 1 or 4 is an alteration at one or more positions selected from the group consisting of K74, R40, Q53, K55, F82, and L79.
  • ActRIIB polypeptides and ActRIIB-based GDF trap polypeptides are contemplated, such as the following.
  • An ActRIIB polypeptide or ActRIIB-based GDF trap polypeptide comprising an amino acid sequence that is at least 80% ( e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO: 1 or 4, and wherein the protein comprises at least one N-X-S/T sequence at a position other than an endogenous N-X-S/T sequence of ActRIIB, and at a position outside of the ligand binding pocket.
  • Any of the above ActRIIB polypeptides or ActRIIB-based GDF trap polypeptides, or fusion proteins thereof, may be produced as a homodimer.
  • Any of the above ActRIIB fusion proteins or ActRIIB-based GDF trap fusion proteins may have a heterologous portion that comprises a constant region from an IgG heavy chain, such as an Fc domain.
  • a preferred ActRIIB polypeptide for use in accordance with the methods disclosed herein, comprises an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NOs: 2, 3, 5, 6, 29, 31, or 49, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing.
  • An ActRIIB polypeptide may include a functional fragment of a natural ActRIIB polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 2, 3, 5, 6, 29, 31, or 49 or a sequence of SEQ ID NO: 2 or 5, lacking the C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at the N-terminus.
  • a preferred polypeptide will comprise a truncation relative to SEQ ID NO: 2 or 5 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus.
  • a preferred GDF trap for use in accordance with the methods described herein consists of, or consists essentially of, the amino acid sequence of SEQ ID NO:29.
  • ActRIIA polypeptide is one that comprises a polypeptide that starts at amino acid 30 and ends at amino acid 110 of SEQ ID NO:9. Accordingly, ActRIIA polypeptides and ActRIIA-based GDF traps of the present disclosure may comprise, consist, or consist essentially of a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO:9.
  • ActRIIA polypeptides and ActRIIA-based GDF trap polypeptides of the present disclosure comprise, consists, or consist essentially of a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids amino acids 12-82 of SEQ ID NO:9 optionally beginning at a position ranging from 1-5 ( e.g., 1, 2, 3, 4, or 5) or 3-5 ( e.g., 3, 4, or 5) and ending at a position ranging from 110-116 ( e.g., 110, 111, 112, 113, 114, 115, or 116) or 110-115 ( e.g., 110, 111, 112, 113, 114, or 115) or SEQ ID NO:9, respectively, and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and zero, one or more non-conservative alterations at positions 40, 53, 55, 74, 79
  • any of the foregoing ActRIIA polypeptides or ActRIIA-based GDF trap polypeptides may be produced as a homodimer. Any of the foregoing ActRIIA polypeptides or ActRIIA-based GDF trap polypeptides may further comprise a heterologous portion that comprises a constant region from an IgG heavy chain, such as an Fc domain. Any of the above ActRIIA polypeptides or ActRIIA-based GDF trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by activin (e.g ., activin A, activin B, activin C, or activin AB) in a cell-based assay.
  • activin e.g ., activin A, activin B, activin C, or activin AB
  • any of the above ActRIIA polypeptides or ActRIIA-based GDF trap polypeptides, including homodimer and/or fusion proteins thereof, may bind to and/or inhibit signaling by GDF11 and/or GDF8 in a cell-based assay.
  • any of the above ActRIIA polypeptides or ActRIIB-based GDF trap polypeptides, including homodimer and/or fusion proteins thereof may bind to and/or inhibit signaling of one or more of activin B, activin C, activin E, GDF7, and Nodal in a cell-based assay.
  • preferred ActRIIA polypeptides and ActRIIA-based GDF-trap polypeptides for use in accordance with the methods disclosed herein, comprise an amino acid sequence that comprises, consists of, or consists essentially of, the amino acid sequence of SEQ ID NOs: 9, 10, 22, 26, or 28, and polypeptides that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any of the foregoing.
  • An ActRIIA polypeptide or ActRIIA-based GDF-trap polypeptide may include a functional fragment of a natural ActRIIA polypeptide, such as one comprising at least 10, 20 or 30 amino acids of a sequence selected from SEQ ID NOs: 9, 10, 22, 26, or 28 or a sequence of SEQ ID NO:10, lacking the C-terminal 1, 2, 3, 4, 5 or 10 to 15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at the N-terminus.
  • a preferred polypeptide will comprise a truncation relative to SEQ ID NO:10 of between 2 and 5 amino acids at the N-terminus and no more than 3 amino acids at the C-terminus.
  • a preferred ActRIIA polypeptide for use in the methods described herein consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 26 or 28.
  • An ActRII polypeptide e.g . an ActRIIA or ActRIIB polypeptide
  • GDF trap polypeptide of the disclosure may include one or more alterations (e.g ., amino acid additions, deletions, substitutions, or combinations thereof) in the amino acid sequence of an ActRII polypeptide ( e.g ., in the ligand-binding domain) relative to a naturally occurring ActRII polypeptide.
  • the alteration in the amino acid sequence may, for example, alter glycosylation of the polypeptide when produced in a mammalian, insect, or other eukaryotic cell or alter proteolytic cleavage of the polypeptide relative to the naturally occurring ActRII polypeptide.
  • ActRII polypeptides e.g . an ActRIIA or ActRIIB polypeptides
  • GDF trap polypeptides of the disclosure comprise one or more modified amino acid residues selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent.
  • an ActRII polypeptide e.g . an ActRIIA or ActRIIB polypeptide
  • GDF trap polypeptide of the disclosure may be a fusion protein that has, as one domain, an ActRII polypeptide or GDF trap polypeptide (e.g., a ligand-binding domain of an ActRII receptor, optionally with one or more sequence variations) and one or more additional heterologous domains that provide a desirable property, such as improved pharmacokinetics, easier purification, targeting to particular tissues, etc.
  • a domain of a fusion protein may enhance one or more of in vivo stability, in vivo half-life, uptake/administration, tissue localization or distribution, formation of protein complexes, multimerization of the fusion protein, and/or purification.
  • ActRII polypeptide and GDF trap fusion proteins may include an immunoglobulin Fc domain (wild-type or mutant) or a serum albumin.
  • an ActRII polypeptide and GDF trap fusion protein comprises a relatively unstructured linker positioned between the Fc domain and the ActRII or GDF trap domain.
  • This unstructured linker may correspond to the roughly 15 amino acid unstructured region at the C-terminal end of the extracellular domain of ActRII or GDF trap (the "tail"), or it may be an artificial sequence of between 3 and 5, 15, 20, 30, 50 or more amino acids that are relatively free of secondary structure.
  • a linker may be rich in glycine and proline residues and may, for example, contain repeating sequences of threonine/serine and glycines [ e.g ., TG 4 (SEQ ID NO:52), TG 3 (SEQ ID NO:53), or SG 4 (SEQ ID NO:54) singlets or repeats] or a series of three glycines.
  • a fusion protein may include a purification subsequence, such as an epitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.
  • an ActRII fusion protein or GDF trap fusion comprises a leader sequence.
  • the leader sequence may be a native ActRII leader sequence (e.g ., a native ActRIIA or ActRIIB leader sequence) or a heterologous leader sequence.
  • the leader sequence is a tissue plasminogen activator (TPA) leader sequence.
  • TPA tissue plasminogen activator
  • an ActRII fusion protein or GDF trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C.
  • the B portion is an N- and C-terminally truncated ActRII or GDF trap polypeptide as described herein.
  • the A and C portions may be independently zero, one, or more than one amino acids, and both A and C portions are heterologous to B.
  • the A and/or C portions may be attached to the B portion via a linker sequence.
  • ActRII polypeptides e.g ., ActRIIA and ActRIIB polypeptides
  • GDF trap polypeptides including variants and fusion proteins thereof, to be used in accordance with the methods disclosed herein bind to one or more ActRIIB ligands (e.g ., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, and/or Nodal) with a Kd less than 10 micromolar, less than 1 micromolar, less than 100 nanomolar, less than 10 nanomolar, or less than 1 nanomolar.
  • ActRIIB ligands e.g ., activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, BMP6, BMP7, and/or Nodal
  • ActRII polypeptides or GDF trap polypeptides inhibit ActRII signaling, such as ActRIIA and/or ActRIIB intracellular signal transduction events triggered by an ActRII ligand (e.g ., SMAD 2/3 and/or SMAD 1/5/8 signaling).
  • ActRII signaling such as ActRIIA and/or ActRIIB intracellular signal transduction events triggered by an ActRII ligand (e.g ., SMAD 2/3 and/or SMAD 1/5/8 signaling).
  • the disclosure provides pharmaceutical preparations comprising an ActRII antagonist of the present disclosure (e.g ., an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap polypeptide) and a pharmaceutically acceptable carrier.
  • a pharmaceutical preparation may also include one or more additional compounds such as a compound that is used to treat a disorder or condition described herein (e.g., an addition compound that increases red blood cell levels and/or hemoglobin levels in a subject in need thereof, treats or prevents anemia in a subject in need thereof, treats sickle-cell disease is a subject in need thereof, treat or prevents one or more complications of sickle-cell disease is a subject in need thereof).
  • a pharmaceutical preparation of the disclosure is substantially pyrogen-free.
  • an ActRIIA polypeptide, an ActRIIB polypeptide, or a GDF trap polypeptide be expressed in a mammalian cell line that mediates suitably natural glycosylation of the polypeptide so as to diminish the likelihood of an unfavorable immune response in a patient.
  • Human and CHO cell lines have been used successfully, and it is expected that other common mammalian expression vectors will be useful.
  • preferable ActRIIA polypeptides, ActRIIB polypeptides, and GDF trap polypeptides are glycosylated and have a glycosylation pattern that is obtainable from a mammalian cell, preferably a CHO cell.
  • the disclosure provides packaged pharmaceuticals comprising a pharmaceutical preparation described herein and labeled for use in one or more of increasing red blood cell levels and/or hemoglobin in a mammal (preferably a human), treating or preventing anemia in a mammal (preferably a human), treating sickle-cell disease in a mamal (preferably a human), and/or treating or preventing one or more complications of sickle-cell disease (e.g ., anemia, vaso-occlusive crisis, etc. ) in a mammal (preferably a human).
  • a mammal preferably a human
  • the disclosure provides nucleic acids encoding an ActRII polypeptide (e.g ., an ActRIIA or ActRIIB polypeptide) or GDF trap polypeptide.
  • An isolated polynucleotide may comprise a coding sequence for a soluble ActRII polypeptide or GDF trap polypeptide, such as described herein.
  • an isolated nucleic acid may include a sequence coding for an ActRII polypeptide or GDF trap comprising an extracellular domain (e.g ., ligand-binding domain) of an ActRII polypeptide having one or more sequence variations and a sequence that would code for part or all of the transmembrane domain and/or the cytoplasmic domain of an ActRII polypeptide, but for a stop codon positioned within the transmembrane domain or the cytoplasmic domain, or positioned between the extracellular domain and the transmembrane domain or cytoplasmic domain.
  • an extracellular domain e.g ., ligand-binding domain
  • an isolated polynucleotide coding for a GDF trap may comprise a full-length ActRII polynucleotide sequence such as SEQ ID NO: 1, 4, or 9 or having one or more variations, or a partially truncated version, said isolated polynucleotide further comprising a transcription termination codon at least six hundred nucleotides before the 3'-terminus or otherwise positioned such that translation of the polynucleotide gives rise to an extracellular domain optionally fused to a truncated portion of a full-length ActRII.
  • Nucleic acids disclosed herein may be operably linked to a promoter for expression, and the disclosure provides cells transformed with such recombinant polynucleotides.
  • the cell is a mammalian cell, such as a CHO cell.
  • the disclosure provides methods for making an ActRII polypeptide or a GDF trap.
  • a method may include expressing any of the nucleic acids disclosed herein (e.g., SEQ ID NO: 8, 13, 27, 32, 39, 42, 46, or 48) in a suitable cell, such as a Chinese hamster ovary (CHO) cell.
  • a suitable cell such as a Chinese hamster ovary (CHO) cell.
  • Such a method may comprise: a) culturing a cell under conditions suitable for expression of the GDF trap polypeptide, wherein said cell is transformed with a GDF trap expression construct; and b) recovering the GDF trap polypeptide so expressed.
  • GDF trap polypeptides may be recovered as crude, partially purified or highly purified fractions using any of the well-known techniques for obtaining protein from cell cultures.
  • the present disclosure relates to an antibody, or combination of antibodies, that antagonize ActRII activity (e.g ., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling).
  • ActRII activity e.g ., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling.
  • the disclosure provides methods of using an antibody ActRII antagonist, or combination of antibody ActRII antagonists, to, e.g ., increase red blood cell levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, treat or prevent sickle-cell disease in a subject in need thereof, and/or treat or prevent one or more complication of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneury
  • a preferred antibody ActRII antagonist of the disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of at least GDF11 (e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • GDF11 e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • the antibody, or combination of antibodies further binds to and/or inhibits activity of GDF8 (e.g ., GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly in the case of a multispecific antibody that has binding affinity for both GDF11 and GDF8 or in the context of a combination of one or more anti-GDF11 antibody and one or more anti-GDF8 antibody.
  • an antibody, or combination of antibodies, of the disclosure does not substantially bind to and/or inhibit activity of activin A (e.g ., activin A-mediated activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • an antibody, or combination of antibodies, of the disclosure that binds to and/or inhibits the activity of GDF11 and/or GDF8 further binds to and/or inhibits activity of one of more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal (e.g., activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the case of a multispecific antibody that has binding affinity for multiple ActRII ligands or in the context of a combination multiple antibodies - each having binding affinity for a different ActRII ligand.
  • activin A activin B
  • activin AB activin C
  • activin E activin E
  • BMP6, BMP7 BMP7
  • Nodal e.g., activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signal
  • the disclosure demonstrates that ActRII antagonists may be used in combination ( e.g ., administered at the same time or different times, but generally in such a manner as to achieve overlapping pharmacological effects) with EPO receptor activators to increase red blood cell levels (erythropoiesis) or treat anemia in patients in need thereof.
  • a GDF trap can be administered in combination with an EPO receptor activator to synergistically increase formation of red blood cells in a patient, particularly in sickle-cell patients.
  • the effect of this combined treatment can be significantly greater than the sum of the effects of the ActRII antagonists and the EPO receptor activator when administered separately at their respective doses.
  • this synergism may be advantageous since it enables target levels of red blood cells to be attained with lower doses of an EPO receptor activator, thereby avoiding potential adverse effects or other problems associated with higher levels of EPO receptor activation.
  • the methods of the present disclosure e.g ., methods of increasing red blood cell levels and/or hemoglobin in a subject in need thereof, treating or preventing anemia in a subject in need thereof, treating sickle-cell disease in a subject in need thereof, and/or treating or preventing one or more complications of sickle-cell disease in a subject in need thereof
  • ActRII antagonists e.g ., ActRIIA polypeptides, ActRIIB polypeptides, and/or GDF trap polypeptides
  • EPO receptor activator may stimulate erythropoiesis by directly contacting and activating EPO receptor.
  • the EPO receptor activator is one of a class of compounds based on the 165 amino-acid sequence of native EPO and generally known as erythropoiesis-stimulating agents (ESAs), examples of which are epoetin alfa, epoetin beta (NeoRecormonTM), epoetin delta (DynepoTM), and epoetin omega.
  • ESAs erythropoiesis-stimulating agents
  • ESAs include synthetic EPO proteins (SEPs) and EPO derivatives with nonpeptidic modifications conferring desirable pharmacokinetic properties (lengthened circulating half-life), examples of which are darbepoetin alfa (AranespTM) and methoxy-polyethylene-glycol epoetin beta (MiceraTM).
  • an EPO receptor activator may be an EPO receptor agonist that does not incorporate the EPO polypeptide backbone or is not generally classified as an ESA.
  • EPO receptor agonists may include, but are not limited to, peptidic and nonpeptidic mimetics of EPO, agonistic antibodies targeting EPO receptor, fusion proteins comprising an EPO mimetic domain, and erythropoietin receptor extended-duration limited agonists (EREDLA).
  • EREDLA erythropoietin receptor extended-duration limited agonists
  • an EPO receptor activator may stimulate erythropoiesis indirectly, without contacting EPO receptor itself, by enhancing production of endogenous EPO.
  • hypoxia-inducible transcription factors HIFs
  • HIFs hypoxia-inducible transcription factors
  • the disclosure provides increased erythropoiesis in a patient by combined treatment with a GDF trap and an indirect EPO receptor activator with HIF stabilizing properties, such as a prolyl hydroxylase inhibitor.
  • ActRII antagonists may also be used for treating or preventing other disorders and conditions such as promoting muscle growth and/or treating or preventing a muscle-related disorder, promoting bone growth and/or treating or preventing a bone-related disorder, treating or preventing cancer (particularly multiple myeloma and/or breast cancer) [see, e.g., U.S. Patent Nos: 7,612,041 ; 8,173,601 ; 7,842,663 as well as U.S. Patent Application Publication No. U.S. 2009/0074768 ].
  • a GDF trap polypeptide when administering a GDF trap polypeptide for these other therapeutic indications, it may be desirable to monitor the effects on red blood cells during administration of the ActRII antagonist, or to determine or adjust the dosing of the ActRII antagonist, in order to reduce undesired effects on red blood cells. For example, increases in red blood cell levels, hemoglobin levels, or hematocrit levels may cause increases in blood pressure.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist. In other aspects, the disclosure provides a method for preventing sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:10.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:10.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:22.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:22.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:36.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:36.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:37.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:37.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:44.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:44.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 29-109 of SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 29-109 of SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 25-131 of SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 25-131 of SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute or chronic renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage, intraventricular hemorrhage, peripheral retinal ischemia, proliferative sickle retinopathy, vitreous hemorrhage, and/or priapism) in a subject
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is anemia. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is anemia crisis. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is splenomegaly.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is pain crisis. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is chest syndrome. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is acute chest syndrome.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is blood transfusion requirement. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is organ damage. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is pain medicine requirement.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is splenic sequestration crises. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is hyperhemolytic crisis.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is vaso-occlusion. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is vaso-occlusion crisis.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is acute myocardial infarction. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is sickle-cell chronic lung disease.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is thromboemboli. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is hepatic failure.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is hepatomegaly. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is hepatic sequestration. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is iron overload.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is splenic infarction. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is acute or chronic renal failure.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is pyelonephritis. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is aneurysm.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is ischemic stroke. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is intraparenchymal hemorrhage.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is subarachnoid hemorrhage. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is intraventricular hemorrhage.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is peripheral retinal ischemia. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is proliferative sickle retinopathy.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is vitreous hemorrhage. In some embodiments, the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof an ActRII antagonist, wherein the complication of sickle-cell disease is priapism.
  • the disclosure provides a method for preventing a complication of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage, intraventricular hemorrhage, peripheral retinal ischemia, proliferative sickle retinopathy, vitreous hemorrhage, and/or priapism) in
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:10.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:10.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:10, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:11, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:22.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:22.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:22, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:36.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:36.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:36, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:37.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:37.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:37, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the amino acid sequence of SEQ ID NO:44.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the amino acid sequence of SEQ ID NO:44.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the amino acid sequence of SEQ ID NO:44, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 29-109 of SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 29-109 of SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 25-131 of SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 25-131 of SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to activin ( e.g ., activin A and/or activin B).
  • activin e.g ., activin A and/or activin B
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% ( e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF11 and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin ( e.g ., activin A and/or activin B).
  • a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide binds to GDF8, GDF11, and activin (e.g ., activin A and/or activin B).
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 29-109 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF8 and GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide consisting of the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF11.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF8.
  • the disclosure provides a method for treating a complication of sickle-cell disease in a subject comprising administering to a subject in need thereof a polypeptide comprising an amino acid sequence that is at least 80% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the sequence of amino acids 25-131 of SEQ ID NO:1, wherein the polypeptide comprises an acidic amino acid at position 79 with respect to SEQ ID NO:1, and wherein the polypeptide binds to GDF8 and GDF11.
  • TGF-beta The transforming growth factor-beta (TGF-beta) superfamily contains a variety of growth factors that share common sequence elements and structural motifs. These proteins are known to exert biological effects on a large variety of cell types in both vertebrates and invertebrates. Members of the superfamily perform important functions during embryonic development in pattern formation and tissue specification and can influence a variety of differentiation processes, including adipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis, neurogenesis, and epithelial cell differentiation. By manipulating the activity of a member of the TGF-beta family, it is often possible to cause significant physiological changes in an organism.
  • the Piedmontese and Belgian Blue cattle breeds carry a loss-of-function mutation in the GDF8 (also called myostatin) gene that causes a marked increase in muscle mass [see, e.g., Grobet et al. (1997) Nat Genet. 17(1):71-4 ].
  • GDF8 also called myostatin
  • inactive alleles of GDF8 are associated with increased muscle mass and, reportedly, exceptional strength [see, e.g., Schuelke et al. (2004) N Engl J Med, 350:2682-8 ].
  • TGF- ⁇ signals are mediated by heteromeric complexes of type I and type II serine/threonine kinase receptors, which phosphorylate and activate downstream SMAD proteins (e.g., SMAD proteins 1, 2, 3, 5, and 8) upon ligand stimulation [see, e.g., Massagué (2000) Nat. Rev. Mol. Cell Biol. 1:169-178 ].
  • SMAD proteins e.g., SMAD proteins 1, 2, 3, 5, and 8
  • SMAD proteins e.g., SMAD proteins 1, 2, 3, 5, and 8
  • Type I and type II receptors are transmembrane proteins, composed of a ligand-binding extracellular domain with cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine specificity.
  • Type I receptors are essential for signaling.
  • Type II receptors are required for binding ligands and for activation of type I receptors.
  • ActRII Two related type II receptors (ActRII), ActRIIA and ActRIIB, have been identified as the type II receptors for activins [see, e.g ., Mathews and Vale (1991) Cell 65:973-982 ; and Attisano et al. (1992) Cell 68: 97-108 ].
  • ActRIIA and ActRIIB can biochemically interact with several other TGF- ⁇ family proteins including, for example, BMP6, BMP7, Nodal, GDF8, and GDF11 [see, e.g ., Yamashita et al. (1995) J. Cell Biol. 130:217-226 ; Lee and McPherron (2001) Proc. Natl.
  • ALK4 is the primary type I receptor for activins, particularly for activin A, and ALK-7 may serve as a receptor for other activins as well, particularly for activin B.
  • the present disclosure relates to antagonizing a ligand of an ActRII receptor (also referred to as an ActRII ligand) with one or more inhibitor agents disclosed herein, particularly inhibitor agents that can antagonize one or more of activin A, activin B, activin C, activin E, GDF11, and/or GDF8.
  • Activins are dimeric polypeptide growth factors that belong to the TGF-beta superfamily. There are three principal activin forms (A, B, and AB) that are homo/heterodimers of two closely related ⁇ subunits ( ⁇ A ⁇ A , ⁇ B ⁇ B , and ⁇ A ⁇ B , respectively).
  • the human genome also encodes an activin C and an activin E, which are primarily expressed in the liver, and heterodimeric forms containing ⁇ C or ⁇ E are also known.
  • activins are unique and multifunctional factors that can stimulate hormone production in ovarian and placental cells, support neuronal cell survival, influence cell-cycle progress positively or negatively depending on cell type, and induce mesodermal differentiation at least in amphibian embryos [ DePaolo et al. (1991) Proc Soc Ep Biol Med. 198:500-512 ; Dyson et al. (1997) Curr Biol. 7:81-84 ; and Woodruff (1998) Biochem Pharmacol. 55:953-963 ]. Moreover, erythroid differentiation factor (EDF) isolated from the stimulated human monocytic leukemic cells was found to be identical to activin A [ Murata et al.
  • EDF erythroid differentiation factor
  • activin A promotes erythropoiesis in the bone marrow.
  • activin signaling is antagonized by its related heterodimer, inhibin.
  • FSH follicle-stimulating hormone
  • Other proteins that may regulate activin bioactivity and/or bind to activin include follistatin (FS), follistatin-related protein (FSRP, also known as FLRG or FSTL3), and ⁇ 2 -macroglobulin.
  • agents that bind to "activin A” are agents that specifically bind to the ⁇ A subunit, whether in the context of an isolated ⁇ A subunit or as a dimeric complex (e.g., a ⁇ A ⁇ A homodimer or a ⁇ A ⁇ B heterodimer).
  • agents that bind to "activin A” are specific for epitopes present within the ⁇ A subunit, but do not bind to epitopes present within the non- ⁇ A subunit of the complex (e.g., the ⁇ B subunit of the complex).
  • agents disclosed herein that antagonize (inhibit) "activin A” are agents that inhibit one or more activities as mediated by a ⁇ A subunit, whether in the context of an isolated ⁇ A subunit or as a dimeric complex (e.g ., a ⁇ A ⁇ A homodimer or a ⁇ A ⁇ B heterodimer).
  • agents that inhibit "activin A” are agents that specifically inhibit one or more activities of the ⁇ A subunit, but do not inhibit the activity of the non- ⁇ A subunit of the complex (e.g ., the ⁇ B subunit of the complex).
  • Agents disclosed herein that antagonize "activin AB” are agents that inhibit one or more activities as mediated by the ⁇ A subunit and one or more activities as mediated by the ⁇ B subunit.
  • Nodal proteins have functions in mesoderm and endoderm induction and formation, as well as subsequent organization of axial structures such as heart and stomach in early embryogenesis. It has been demonstrated that dorsal tissue in a developing vertebrate embryo contributes predominantly to the axial structures of the notochord and pre-chordal plate while it recruits surrounding cells to form non-axial embryonic structures. Nodal appears to signal through both type I and type II receptors and intracellular effectors known as SMAD proteins. Studies support the idea that ActRIIA and ActRIIB serve as type II receptors for Nodal [see, e.g., Sakuma et al. (2002) Genes Cells. 2002, 7:401-12 ].
  • Nodal ligands interact with their co-factors (e.g ., cripto) to activate activin type I and type II receptors, which phosphorylate SMAD2.
  • Nodal proteins are implicated in many events critical to the early vertebrate embryo, including mesoderm formation, anterior patterning, and left-right axis specification.
  • Experimental evidence has demonstrated that Nodal signaling activates pAR3-Lux, a luciferase reporter previously shown to respond specifically to activin and TGF-beta.
  • Nodal is unable to induce pTlx2-Lux, a reporter specifically responsive to bone morphogenetic proteins.
  • GDF8 Growth and differentiation factor-8
  • GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highly expressed in the developing and adult skeletal muscle. The GDF8 null mutation in transgenic mice is characterized by a marked hypertrophy and hyperplasia of the skeletal muscle [ McPherron et al., Nature (1997) 387:83-90 ]. Similar increases in skeletal muscle mass are evident in naturally occurring mutations of GDF8 in cattle [see, e.g., Ashmore et al. (1974) Growth, 38:501-507 ; Swatland and Kieffer (1994) J. Anim. Sci. 38:752-757 ; McPherron and Lee (1997) Proc. Natl.
  • GDF8 can modulate the production of muscle-specific enzymes (e.g., creatine kinase) and modulate myoblast cell proliferation [see, e.g. international patent application publication no.
  • the GDF8 propeptide can noncovalently bind to the mature GDF8 domain dimer, inactivating its biological activity [see, e.g., Miyazono et al. (1988) J. Biol. Chem., 263: 6407-6415 ; Wakefield et al. (1988) J. Biol. Chem., 263: 7646-7654 ; and Brown et al. (1990) Growth Factors, 3: 35-43 ].
  • Other proteins which bind to GDF8 or structurally related proteins and inhibit their biological activity include follistatin, and potentially, follistatin-related proteins [see, e.g., Gamer et al. (1999) Dev. Biol., 208: 222-232 ].
  • GDF11 Growth and differentiation factor-11
  • BMP11 is a secreted protein [ McPherron et al. (1999) Nat. Genet. 22: 260-264 ]. GDF11 is expressed in the tail bud, limb bud, maxillary and mandibular arches, and dorsal root ganglia during mouse development [see, e.g., Nakashima et al. (1999) Mech. Dev. 80: 185-189 ]. GDF11 plays a unique role in patterning both mesodermal and neural tissues [see, e.g., Gamer et al. (1999) Dev Biol., 208:222-32 ].
  • GDF11 was shown to be a negative regulator of chondrogenesis and myogenesis in developing chick limb [see, e.g., Gamer et al. (2001) Dev Biol. 229:407-20 ].
  • the expression of GDF11 in muscle also suggests its role in regulating muscle growth in a similar way to GDF8.
  • the expression of GDF11 in brain suggests that GDF11 may also possess activities that relate to the function of the nervous system.
  • GDF11 was found to inhibit neurogenesis in the olfactory epithelium [see, e.g., Wu et al. (2003) Neuron. 37:197-207 ].
  • Bone morphogenetic protein also called osteogenic protein-1 (OP-1)
  • BMP7 also called osteogenic protein-1 (OP-1)
  • BMP7 regulates a wide array of physiological processes.
  • BMP7 may be the osteoinductive factor responsible for the phenomenon of epithelial osteogenesis. It is also found that BMP7 plays a role in calcium regulation and bone homeostasis.
  • BMP7 binds to type II receptors, ActRIIA and ActRIIB.
  • BMP7 and activin recruit distinct type I receptors into heteromeric receptor complexes.
  • the major BMP7 type I receptor observed was ALK2, while activin bound exclusively to ALK4 (ActRIIB).
  • BMP7 and activin elicited distinct biological responses and activated different SMAD pathways [see, e.g ., Macias-Silva et al. (1998) J Biol Chem. 273:25628-36 ].
  • ActRII polypeptides can be used to increase red blood cell levels in vivo.
  • a GDF trap polypeptide specifically a variant ActRIIB polypeptide
  • This GDF trap is characterized, in part, by substantial loss of binding affinity for activin A, and therefore significantly diminished capacity to antagonize activin A activity, but retains near wild-type levels of binding and inhibition of GDF11.
  • the GDF trap is more effective at increasing red blood cell levels compared to the wild-type ActRII polypeptide and has beneficial effects in a variety of models for anemia and sickle-cell disease.
  • a GDF trap polypeptide can be used to improve red blood cell morphology, increase red blood cell levels, increase the oxygen carrying capacity of hemoglobin, and prevent tissue damage due to vascular congestion and hypoxia in sickle-cell patients, which indicates a much broader use for ActRII antagonists in the treatment of sickle-cell disease beyond the alleviation of anemia.
  • hematopoiesis is a complex process, regulated by a variety of factors, including erythropoietin, G-CSF, and iron homeostasis.
  • increase red blood cell levels and “promote red blood cell formation” refer to clinically observable metrics, such as hematocrit, red blood cell counts, and hemoglobin measurements, and are intended to be neutral as to the mechanism by which such changes occur.
  • the data of the present disclosure therefore indicate that the observed biological activity of an ActRII polypeptide, with respect to red blood cell levels, is not dependent on activin A inhibition.
  • the unmodified ActRIIB polypeptide, which retains activin A binding still demonstrates the capacity to increase red blood cells in vivo.
  • an ActRIIB or ActRIIA polypeptide that retains activin A inhibition may be more desirable in some applications, in comparison to a GDF trap having diminished binding affinity for activin A, where more modest gains in red blood cell levels are desirable and/or where some level of off-target activity is acceptable (or even desirable).
  • the methods of the present disclosure are directed to the use of one or more ActRII antagonist agents described herein, optionally in combination with one or more supportive therapies, to increase red blood cell formation in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, to treat sickle-cell disease in a subject in need thereof, and to treat or prevent one or more complications of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneur
  • sickle-cell disease
  • the ActRII antagonist agents described herein may be used in combination with an EPO receptor activator or in patients that have failed treatment with an EPO receptor activator.
  • EPO is a glycoprotein hormone involved in the growth and maturation of erythroid progenitor cells into erythrocytes. EPO is produced by the liver during fetal life and by the kidney in adults. Decreased production of EPO, which commonly occurs in adults as a consequence of renal failure, leads to anemia. EPO has been produced by genetic engineering techniques based on expression and secretion of the protein from a host cell transfected with the EPO gene. Administration of such recombinant EPO has been effective in the treatment of anemia. For example, Eschbach et al. (1987, N Engl J Med 316:73 ) describe the use of EPO to correct anemia caused by chronic renal failure.
  • EPO Effects of EPO are mediated through its binding to, and activation of, a cell surface receptor belonging to the cytokine receptor superfamily and designated the EPO receptor.
  • the human and murine EPO receptors have been cloned and expressed [see, e.g ., D'Andrea et al. (1989) Cell 57:277 ; Jones et al. (1990) Blood 76:31 ; Winkelman et al. (1990) Blood 76:24 ; and U.S. Pat. No. 5,278,065 ].
  • the human EPO receptor gene encodes a 483-aminoacid transmembrane protein comprising an extracellular domain of approximately 224 amino acids and exhibits approximately 82% amino acid sequence identity with the murine EPO receptor (see, e.g., U.S. Pat. No. 6,319,499 ).
  • this form is thought to contain the main EPO binding determinant and is referred to as the EPO receptor.
  • the EPO receptor is thought to dimerize upon agonist binding. Nevertheless, the detailed structure of the EPO receptor, which may be a multimeric complex, and its specific mechanism of activation are not completely understood (see, e.g., U.S. Pat. No. 6,319,499 ).
  • EPO receptor Activation of the EPO receptor results in several biological effects. These include increased proliferation of immature erythroblasts, increased differentiation of immature erythroblasts, and decreased apoptosis in erythroid progenitor cells [see, e.g., Liboi et al. (1993) Proc Natl Acad Sci USA 90:11351-11355 ; Koury et al. (1990) Science 248:378-381 ].
  • the EPO receptor signal transduction pathways mediating proliferation and differentiation appear to be distinct [see, e.g., Noguchi et al. (1988) Mol Cell Biol 8:2604 ; Patel et al.
  • EPO receptor activators include small molecule erythropoiesis-stimulating agents (ESAs) as well as EPO-based compounds.
  • ESAs small molecule erythropoiesis-stimulating agents
  • An example of the former is a dimeric peptide-based agonist covalently linked to polyethylene glycol (proprietary names HematideTM and Omontys®), which has shown erythropoiesis-stimulating properties in healthy volunteers and in patients with both chronic kidney disease and endogenous anti-EPO antibodies [see, e.g., Stead et al. (2006) Blood 108:1830-1834 ; and Macdougall et al. (2009) N Engl J Med 361:1848-1855 ].
  • Other examples include nonpeptide-based ESAs [see, e.g., Qureshi et al. (1999) Proc Natl Acad Sci USA 96:12156-12161 ].
  • EPO receptor activators also include compounds that stimulate erythropoiesis indirectly, without contacting EPO receptor itself, by enhancing production of endogenous EPO.
  • hypoxia-inducible transcription factors HIFs
  • HIF prolyl hydroxylase enzymes are endogenous stimulators of EPO gene expression that are suppressed (destabilized) under normoxic conditions by cellular regulatory mechanisms. Therefore, inhibitors of HIF prolyl hydroxylase enzymes are being investigated for EPO-inducing activity in vivo.
  • Other indirect activators of EPO receptor include inhibitors of GATA-2 transcription factor [see, e.g., Nakano et al.
  • HCP hemopoietic cell phosphatase
  • sequence similarity in all its grammatical forms, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences that may or may not share a common evolutionary origin.
  • homologous when modified with an adverb such as “highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • Percent (%) sequence identity with respect to a reference polypeptide (or nucleotide) sequence is defined as the percentage of amino acid residues (or nucleic acids) in a candidate sequence that are identical to the amino acid residues (or nucleic acids) in the reference polypeptide (nucleotide) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software.
  • ALIGN-2 sequence comparison computer program
  • the ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code.
  • the ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.
  • does not substantially bind to X is intended to mean that an agent has a K D that is greater than about 10 -7 , 10 -6 , 10 -5 , 10 -4 or greater ( e.g., no detectable binding by the assay used to determine the K D ) for "X".
  • ActRII antagonists (inhibitors) of ActRII
  • antagonists e.g., antagonist of ActRIIA and/or ActRIIB SMAD 2/3 and/or SMAD 1/5/8 signaling
  • ActRII antagonists are shown herein to be effective in treating various anemias as well as various complications (e.g., disorders/conditions) of sickle-cell disease.
  • the present disclosure provides, in part, various ActRII antagonist agents that can be used, alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive therapies [ e.g., treatment with hydroxyurea, blood transfusion, iron chelation therapy, and/or pain management (e.g ., treatment with one or more of opioid analgesic agents, non-steroidal anti-inflammatory drugs, and/or corticosteroids)], to increase red blood cell levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, treat sickle cell disease in a subject in need thereof, and/or treat or prevent one or more complication of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-oc
  • preferred ActRII antagonists to be used in accordance with the methods disclosed herein are GDF-ActRII antagonists (e.g ., antagonists of GDF-mediated ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly GDF11- and/or GDF8-mediated ActRII signaling.
  • preferred ActRII antagonists of the present disclosure are soluble ActRII polypeptides (e.g ., soluble ActRIIA and ActRIIB polypeptides) and GDF trap polypeptides, such as ActRIIA-Fc fusion proteins, ActRIIB-Fc fusion proteins, and GDF trap-Fc fusion proteins.
  • GDF11 and/or GDF8 inhibition may be an indicator of the tendency of an agent to inhibit the activities of a spectrum of additional agents, including, perhaps, other members of the TGF-beta superfamily (e.g ., activin B, activin C, activin E, BMP6, BMP7, and/or Nodal) and such collective inhibition may lead to the desired effect on, e.g ., hematopoiesis]
  • GDF-ActRII antagonist are expected to be useful including, for example, anti-GDFl 1 antibodies; anti-GDF8 antibodies; anti-activin A, B, C, and/or E antibodies, anti-ActRIIA antibodies; anti-ActRIIB antibodies; anti-ActRIIA/
  • GDF-ActRII antagonists of the present disclosure may bind to and/or inhibit the activity (or expression) of other ActRII ligands including, for example, activin A, activin AB, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal.
  • a GDF-ActRII antagonist of the present disclosure may be used in combination with at least one additional ActRII antagonist agent that binds to and/or inhibits the activity (or expression) of one or more additional ActRII ligands including, for example, activin A, activin AB, activin B, activin C, activin E, BMP6, BMP7, and/or Nodal.
  • ActRII antagonists to be used in accordance with the methods disclosed herein do not substantially bind to and/or inhibit activin A (e.g ., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • activin A e.g ., activin A-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • the present disclosure relates to ActRII polypeptides.
  • the disclosure provides methods of using one or more ActRII polypeptides, alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive therapies (e.g ., transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea etc.
  • EPO erythropoiesis stimulating agents
  • supportive therapies e.g ., transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea etc.
  • sickle-cell disease in a subject in need thereof treat or prevent one or more complications of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage,
  • ActRIIB refers to a family of activin receptor type IIB (ActRIIB) proteins from any species and variants derived from such ActRIIB proteins by mutagenesis or other modification. Reference to ActRIIB herein is understood to be a reference to any one of the currently identified forms. Members of the ActRIIB family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
  • ActRIIB polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIB family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIA polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication No. WO 2006/012627 , which is incorporated herein by reference in its entirety. Optionally, ActRIIB polypeptides of the present disclosure can be used to increase red blood cell levels in a subject. Numbering of amino acids for all ActRIIB-related polypeptides described herein is based on the numbering of the human ActRIIB precursor protein sequence provided below (SEQ ID NO:1), unless specifically designated otherwise.
  • the human ActRIIB precursor protein sequence is as follows:
  • the signal peptide is indicated with single underline ; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated with double underline.
  • the processed soluble (extracellular) human ActRIIB polypeptide sequence is as follows:
  • the protein may be produced with an "SGR" sequence at the N-terminus.
  • the C-terminal "tail” of the extracellular domain is indicated by single underline .
  • the sequence with the "tail” deleted is as follows:
  • a form of ActRIIB with an alanine at position 64 of SEQ ID NO: 1 (A64) is also reported in the literature [see, e.g., Hilden et al. (1994) Blood, 83(8): 2163-2170 ].
  • Applicants have ascertained that an ActRIIB-Fc fusion protein comprising an extracellular domain of ActRIIB with the A64 substitution has a relatively low affinity for activin and GDF11.
  • the same ActRIIB-Fc fusion protein with an arginine at position 64 has an affinity for activin and GDF11 in the low nanomolar to high picomolar range. Therefore, sequences with an R64 are used as the "wild-type" reference sequence for human ActRIIB in this disclosure.
  • the signal peptide is indicated by single underline and the extracellular domain is indicated by bold font.
  • the protein may be produced with an "SGR" sequence at the N-terminus.
  • the C-terminal "tail” of the extracellular domain is indicated by single underline .
  • the sequence with the "tail” deleted is as follows:
  • the nucleic acid sequence encoding human ActRIIB precursor protein is shown below (SEQ ID NO: 7), consisting of nucleotides 25-1560 of Genbank Reference Sequence NM_001106.3, which encode amino acids 1-513 of the ActRIIB precursor.
  • the sequence as shown provides an arginine at position 64 and may be modified to provide an alanine instead.
  • the signal sequence is underlined.
  • a nucleic acid sequence encoding processed soluble (extracellular) human ActRIIB polypeptide is as follows (SEQ ID NO: 8). The sequence as shown provides an arginine at position 64, and may be modified to provide an alanine instead.
  • ActRIIA refers to a family of activin receptor type IIA (ActRIIA) proteins from any species and variants derived from such ActRIIA proteins by mutagenesis or other modification. Reference to ActRIIA herein is understood to be a reference to any one of the currently identified forms.
  • ActRIIA family are generally transmembrane proteins, composed of a ligand-binding extracellular domain comprising a cysteine-rich region, a transmembrane domain, and a cytoplasmic domain with predicted serine/threonine kinase activity.
  • ActRIIA polypeptide includes polypeptides comprising any naturally occurring polypeptide of an ActRIIA family member as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity. Examples of such variant ActRIIA polypeptides are provided throughout the present disclosure as well as in International Patent Application Publication No. WO 2006/012627 , which is incorporated herein by reference in its entirety. Optionally, ActRIIA polypeptides of the present disclosure can be used to increase red blood cell levels in a subject. Numbering of amino acids for all ActRIIA-related polypeptides described herein is based on the numbering of the human ActRIIA precursor protein sequence provided below (SEQ ID NO:9), unless specifically designated otherwise.
  • the human ActRIIA precursor protein sequence is as follows:
  • the signal peptide is indicated by single underline ; the extracellular domain is indicated in bold font; and the potential, endogenous N-linked glycosylation sites are indicated by double underline.
  • the processed soluble (extracellular) human ActRIIA polypeptide sequence is as follows:
  • the C-terminal "tail" of the extracellular domain is indicated by single underline.
  • the sequence with the "tail” deleted is as follows:
  • the nucleic acid sequence encoding human ActRIIA precursor protein is shown below (SEQ ID NO: 12), as follows nucleotides 159-1700 of Genbank Reference Sequence NM_001616.4. The signal sequence is underlined.
  • nucleic acid sequence encoding processed soluble (extracellular) human ActRIIA polypeptide is as follows:
  • FIG. 1 An alignment of the amino acid sequences of human ActRIIB soluble extracellular domain and human ActRIIA soluble extracellular domain are illustrated in Figure 1 . This alignment indicates amino acid residues within both receptors that are believed to directly contact ActRII ligands.
  • Figure 2 depicts a multiple-sequence alignment of various vertebrate ActRIIB proteins and human ActRIIA. From these alignments it is possible to predict key amino acid positions within the ligand-binding domain that are important for normal ActRII-ligand binding activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ActRII-ligand binding activities.
  • GDF trap polypeptides also referred to as "GDF traps”
  • GDF traps may be used, for example, alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive therapies (e.g., transfusion of red blood cells, whole blood, iron chelation therapy, treatment with hydroxyurea, etc.
  • EPO erythropoiesis stimulating agents
  • supportive therapies e.g., transfusion of red blood cells, whole blood, iron chelation therapy, treatment with hydroxyurea, etc.
  • sickle-cell disease e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrhage
  • GDF traps of the present disclosure are soluble, variant ActRII polypeptides (e.g ., ActRIIA and ActRIIB polypeptides) that comprise one or more mutations (e.g. , amino acid additions, deletions, substitutions, and combinations thereof) in the extracellular domain (also referred to as the ligand-binding domain) of an ActRII polypeptide (e.g., a "wild-type" ActRII polypeptide) such that the variant ActRII polypeptide has one or more altered ligand-binding activities than the corresponding wild-type ActRII polypeptide.
  • GDF trap polypeptides of the present disclosure retain at least one similar activity as a corresponding wild-type ActRII polypeptide (e.g ., an ActRIIA or ActRIIB polypeptide).
  • a GDF trap may bind to and/or inhibit (e.g . antagonize) the function of one or more ActRII ligands (e.g ., inhibit ActRII ligand-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling pathway).
  • GDF traps of the present disclosure bind to and/or inhibit one or more of activin A, activin B, activin AB, activin C, activin E, Nodal, GDF8, GDF11, BMP6 and/or BMP7).
  • GDF trap polypeptides of the disclosure have elevated binding affinity for one or more specific ActRII ligands (e.g ., GDF8, GDF11, BMP6, Nodal, and/or BMP7). In other embodiments, GDF trap polypeptides of the disclosure have decreased binding affinity for one or more specific ActRII ligands ( e.g ., activin A, activin B, activin AB, activin C, and/or activin E). In still other embodiments, GDF trap polypeptides of the disclosure have elevated binding affinity for one or more specific ActRII ligands and decreased binding affinity for one or more different/other ActRII ligands. Accordingly, the present disclosure provides GDF trap polypeptides that have an altered binding specificity for one or more ActRII ligands.
  • specific ActRII ligands e.g ., GDF8, GDF11, BMP6, Nodal, and/or BMP7.
  • GDF trap polypeptides of the disclosure have decreased binding
  • GDF traps of the present disclosure are designed to preferentially bind to and antagonize GDF11 and/or GDF8 (also known as myostatin), e.g ., in comparision to a wild-type ActRII polypeptide.
  • GDF11 and/or GDF8-binding traps may further bind to and/or antagonize one or more of Nodal, GDF8, GDF11, BMP6 and/or BMP7.
  • GDF11 and/or GDF8-binding traps may further bind to and/or antagonize one or more of activin B, activin C, activin E, Nodal, GDF8, GDF11, BMP6 and/or BMP7.
  • GDF11 and/or GDF8-binding traps may further bind to and/or antagonize one or more of activin A, activin A/B, activin B, activin C, activin E, Nodal, GDF8, GDF11, BMP6 and/or BMP7.
  • GDF traps of the present disclosure have diminished binding affinity for activins (e.g ., activin A, activin A/B, activin B, activin C, activin E), e.g., in comparision to a wild-type ActRII polypeptide.
  • a GDF trap polypeptide of the present disclosure has diminished binding affinity for activin A.
  • the disclosure provides GDF trap polypeptides that preferentially bind to and/or antagonize GDF8/GDF11 relative to activin A.
  • GDF trap polypeptides are more potent activators of erythropoiesis in vivo in comparision to ActRII polypeptides that retain high binding affinity for activin A.
  • these non-activin-A-binding GDF trap polypeptides demonstrate decreased effects on other tissues. Therefore, such GDF traps may be useful for increasing red blood cell levels in a subject while reducing potential off-target effects associated with binding/antagonizing activin A.
  • selective GDF trap polypeptides may be less desirable in some applications wherein more modest gains in red blood cell levels may be needed for therapeutic effect and wherein some level of off-target effect is acceptable (or even desirable).
  • Amino acid residues of the ActRIIB proteins are in the ActRIIB ligand-binding pocket and help mediated binding to its ligands including, for example, activin A, GDF11, and GDF8.
  • GDF trap polypeptides comprising an altered-ligand binding domain (e.g ., a GDF8/GDF11-binding domain) of an ActRIIB receptor which comprises one or more mutations at those amino acid residues.
  • the altered ligand-binding domain can have increased selectivity for a ligand such as GDF11 and/or GDF8 relative to a wild-type ligand-binding domain of an ActRIIB receptor.
  • a ligand such as GDF11 and/or GDF8 relative to a wild-type ligand-binding domain of an ActRIIB receptor.
  • one or more mutations may be selected that increase the selectivity of the altered ligand-binding domain for GDF11 and/or GDF8 over one or more activins (activin A, activin B, activin AB, activin C, and/or activin A), particularly activin A.
  • the altered ligand-binding domain has a ratio of K d for activin binding to K d for GDF11 and/or GDF8 binding that is at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-fold greater relative to the ratio for the wild-type ligand-binding domain.
  • the altered ligand-binding domain has a ratio of IC 50 for inhibiting activin to IC 50 for inhibiting GDF11 and/or GDF8 that is at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-fold greater relative to the wild-type ligand-binding domain.
  • the altered ligand-binding domain inhibits GDF11 and/or GDF8 with an IC 50 at least 2-, 5-, 10-, 20-, 50-, 100- or even 1000-times less than the IC 50 for inhibiting activin.
  • the positively-charged amino acid residue Asp (D80) of the ligand-binding domain of ActRIIB can be mutated to a different amino acid residue to produce a GDF trap polypeptide that preferentially binds to GDF8, but not activin.
  • the D80 residue with respect to SEQ ID NO:1 is changed to an amino acid residue selected from the group consisting of: an uncharged amino acid residue, a negative amino acid residue, and a hydrophobic amino acid residue.
  • the hydrophobic residue L79 of SEQ ID NO:1 can be altered to confer altered activin-GDF11/GDF8 binding properties.
  • an L79P substitution reduces GDF11 binding to a greater extent than activin binding.
  • replacement of L79 with an acidic amino acid [an aspartic acid or glutamic acid; an L79D or an L79E substitution] greatly reduces activin A binding affinity while retaining GDF11 binding affinity.
  • the methods described herein utilize a GDF trap polypeptide which is a variant ActRIIB polypeptide comprising an acidic amino acid (e.g., D or E) at the position corresponding to position 79 of SEQ ID NO: 1, optionally in combination with one or more additional amino acid substitutions, additions, or deletions.
  • the present disclosure relates to ActRII polypeptides (ActRIIA and ActRIIB polypeptides) which are soluble ActRII polypeptides.
  • ActRIIA and ActRIIB polypeptides which are soluble ActRII polypeptides.
  • soluble ActRII polypeptide generally refers to polypeptides comprising an extracellular domain of an ActRII protein.
  • soluble ActRII polypeptide includes any naturally occurring extracellular domain of an ActRII protein as well as any variants thereof (including mutants, fragments, and peptidomimetic forms) that retain a useful activity (e.g ., a GDF trap polypeptide as described herein).
  • soluble ActRII polypeptides comprise a signal sequence in addition to the extracellular domain of an ActRII or GDF trap protein.
  • the signal sequence can be a native signal sequence of an ActRIIA or ActRIIB protein, or a signal sequence from another protein including, for example, a tissue plasminogen activator (TPA) signal sequence or a honey bee melittin (HBM) signal sequence.
  • TPA tissue plasminogen activator
  • HBM honey bee melittin
  • the present disclosure identifies functionally active portions and variants of ActRII polypeptides that can be used as guidance for generating and using ActRIIA polypeptides, ActRIIB polypeptides, and GDF trap polypeptides within the scope of the methods described herein.
  • ActRII proteins have been characterized in the art in terms of structural and functional characteristics, particularly with respect to ligand binding [see, e.g., Attisano et al. (1992) Cell 68(1):97-108 ; Greenwald et al. (1999) Nature Structural Biology 6(1): 18-22 ; Allendorph et al. (2006) PNAS 103(20: 7643-7648 ; Thompson et al. (2003) The EMBO Journal 22(7): 1555-1566 ; and U.S. Patent Nos: 7,709,605 , 7,612,041 , and 7,842,663 ].
  • Attisano et al. showed that a deletion of the proline knot at the C-terminus of the extracellular domain of ActRIIB reduced the affinity of the receptor for activin.
  • An ActRIIB-Fc fusion protein containing amino acids 20-119 of present SEQ ID NO:1, "ActRIIB(20-119)-Fc”, has reduced binding to GDF-11 and activin relative to an ActRIIB(20-134)-Fc, which includes the proline knot region and the complete juxtamembrane domain (see, e.g., U.S. Patent No. 7,842,663 ).
  • an ActRIIB(20-129)-Fc protein retains similar but somewhat reduced activity relative to the wild-type, even though the proline knot region is disrupted.
  • ActRIIB extracellular domains that stop at amino acid 134, 133, 132, 131, 130 and 129 are all expected to be active, but constructs stopping at 134 or 133 may be most active.
  • mutations at any of residues 129-134 (with respect to SEQ ID NO:1) are not expected to alter ligand-binding affinity by large margins.
  • mutations of P129 and P130 (with respect to SEQ ID NO:1) do not substantially decrease ligand binding.
  • an ActRIIB polypeptide or an ActRIIB-based GDF trap polypeptide of the present disclosure may end as early as amino acid 109 (the final cysteine), however, forms ending at or between 109 and 119 ( e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, or 119) are expected to have reduced ligand binding.
  • Amino acid 119 (with respect to present SEQ ID NO:1) is poorly conserved and so is readily altered or truncated.
  • ActRIIB polypeptides and ActRIIB-based GDF traps ending at 128 (with respect to present SEQ ID NO:1) or later should retain ligand binding activity.
  • ActRIIB polypeptides and ActRIIB-based GDF traps ending at or between 119 and 127 will have an intermediate binding ability. Any of these forms may be desirable to use, depending on the clinical or experimental setting.
  • ActRIIB polypeptides and ActRIIB-based GDF traps beginning at position 20, 21, 22, 23, and 24 should retain general ligand-biding activity
  • ActRIIB polypeptides and ActRIIB-based GDF traps beginning at positions 25, 26, 27, 28, and 29 are also expected to retain ligand-biding activity.
  • Data shown herein as well as in, e.g., U.S. Patent No. 7,842,663 demonstrates that, surprisingly, an ActRIIB construct beginning at 22, 23, 24, or 25 will have the most activity.
  • an active portion e.g ., ligand-binding activity of ActRIIB comprises amino acids 29-109 of SEQ ID NO:1. Therefore ActRIIB polypeptides and ActRIIB-based GDF traps of the present disclosure may, for example, comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of ActRIIB beginning at a residue corresponding to amino acids 20-29 ( e.g., beginning at amino acid 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) of SEQ ID NO: 1 and ending at a position corresponding to amino acids 109-134 ( e.g., ending at amino acid 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 13
  • ActRIIB-based GDF trap polypeptides of the present disclosure do not comprise or consist of amino acids 29-109 of SEQ ID NO:1.
  • Other examples include polypeptides that begin at a position from 20-29 ( e.g., position 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) or 21-29 ( e.g., position 21, 22, 23, 24, 25, 26, 27, 28, or 29) and end at a position from 119-134 ( e.g ., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-133 ( e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133), 129-134 ( e.g., 129, 130, 131, 132, 133, or 134), or 129-133 (
  • constructs that begin at a position from 20-24 ( e.g., 20, 21, 22, 23, or 24), 21-24 ( e.g., 21, 22, 23, or 24), or 22-25 ( e.g., 22, 22, 23, or 25) and end at a position from 109-134 ( e.g., 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134), 119-134 ( e.g., 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, or 134) or 129-134 ( e.g., 129, 130, 131, 132, 133, or 134) of SEQ ID NO: 1.
  • 109-134 e.g., 109
  • ActRIIB polypeptides and ActRIIB-based GDF traps comprise a polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acid residues 25-131 of SEQ ID NO: 1.
  • ActRIIB-based GDF trap polypeptides do not comprise or consist of amino acids 25-131 of SEQ ID NO: 1.
  • the disclosure includes the results of an analysis of composite ActRIIB structures, shown in Figure 1 , demonstrating that the ligand-binding pocket is defined, in part, by residues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through F101.
  • residues Y31, N33, N35, L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87, A92, and E94 through F101.
  • R40A, K55A, F82A and mutations at position L79 are conservative mutations at position L79.
  • R40 is a K in Xenopus, indicating that basic amino acids at this position will be tolerated.
  • a general formula for an ActRIIB polypeptide and ActRIIB-based GDF trap polypeptide of the disclosure is one that comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 29-109 of SEQ ID NO: 1, optionally beginning at a position ranging from 20-24 ( e.g., 20, 21, 22, 23, or 24) or 22-25 ( e.g., 22, 23, 24, or 25) and ending at a position ranging from 129-134 ( e.g., 129, 130, 131, 132, 133, or 134), and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand-binding pocket, and zero, one or more non-conservative alterations at positions 40, 53, 55,
  • Sites outside the binding pocket include the amino and carboxy termini of the extracellular domain (as noted above), and positions 42-46 and 65-73 (with respect to SEQ ID NO:1).
  • An asparagine-to-alanine alteration at position 65 actually improves ligand binding in the A64 background, and is thus expected to have no detrimental effect on ligand binding in the R64 background (see, e.g., U.S. Patent No. 7,842,663 ).
  • This change probably eliminates glycosylation at N65 in the A64 background, thus demonstrating that a significant change in this region is likely to be tolerated.
  • R64A change is poorly tolerated
  • R64K is well-tolerated, and thus another basic residue, such as H may be tolerated at position 64 (see, e.g., U.S. Patent No. 7,842,663 ).
  • ActRIIB is well-conserved across nearly all vertebrates, with large stretches of the extracellular domain conserved completely. Many of the ligands that bind to ActRIIB are also highly conserved. Accordingly, comparisons of ActRIIB sequences from various vertebrate organisms provide insights into residues that may be altered. Therefore, an active, human ActRIIB variant polypeptide and ActRIIB-based GDF trap useful in accordance with the presently disclosed methods may include one or more amino acids at corresponding positions from the sequence of another vertebrate ActRIIB, or may include a residue that is similar to that in the human or other vertebrate sequence. The following examples illustrate this approach to defining an active ActRIIB variant.
  • L46 is a valine in Xenopus ActRIIB, and so this position may be altered, and optionally may be altered to another hydrophobic residue, such as V, I or F, or a non-polar residue such as A.
  • E52 is a K in Xenopus, indicating that this site may be tolerant of a wide variety of changes, including polar residues, such as E, D, K, R, H, S, T, P, G, Y and probably A.
  • T93 is a K in Xenopus, indicating that a wide structural variation is tolerated at this position, with polar residues favored, such as S, K, R, E, D, H, G, P, G and Y.
  • F108 is a Y in Xenopus, and therefore Y or other hydrophobic group, such as I, V or L should be tolerated.
  • E111 is K in Xenopus, indicating that charged residues will be tolerated at this position, including D, R, K and H, as well as Q and N.
  • R112 is K in Xenopus, indicating that basic residues are tolerated at this position, including R and H.
  • a at position 119 is relatively poorly conserved, and appears as P in rodents and V in Xenopus, thus essentially any amino acid should be tolerated at this position.
  • N-X-S/T N-linked glycosylation site
  • ActRIIB(R64)-Fc form see, e.g., U.S. Patent No. 7,842,663 .
  • N-X-S/T sequences may be generally introduced at positions outside the ligand binding pocket defined in Figure 1 in ActRIIB polypeptide and ActRIIB-based GDF traps of the present disclosure.
  • Particularly suitable sites for the introduction of non-endogenous N-X-S/T sequences include amino acids 20-29, 20-24, 22-25, 109-134, 120-134 or 129-134 (with respect to SEQ ID NO:1).
  • N-X-S/T sequences may also be introduced into the linker between the ActRIIB sequence and an Fc domain or other fusion component.
  • Such a site may be introduced with minimal effort by introducing an N in the correct position with respect to a pre-existing S or T, or by introducing an S or T at a position corresponding to a pre-existing N.
  • desirable alterations that would create an N-linked glycosylation site are: A24N, R64N, S67N (possibly combined with an N65A alteration), E105N, R112N, G120N, E123N, P129N, A132N, R112S and R112T (with respect to SEQ ID NO:1).
  • an S that is predicted to be glycosylated may be altered to a T without creating an immunogenic site, because of the protection afforded by the glycosylation.
  • any T that is predicted to be glycosylated may be altered to an S.
  • the alterations S67T and S44T (with respect to SEQ ID NO:1) are contemplated.
  • an S26T alteration may be used.
  • an ActRIIB polypeptide and ActRIIB-based GDF trap polypeptide of the present disclosure may be an variant having one or more additional, non-endogenous N-linked glycosylation consensus sequences as described above.
  • the results of the mutagenesis program described herein indicate that there are amino acid positions in ActRIIB that are often beneficial to conserve.
  • these include position 64 (basic amino acid), position 80 (acidic or hydrophobic amino acid), position 78 (hydrophobic, and particularly tryptophan), position 37 (acidic, and particularly aspartic or glutamic acid), position 56 (basic amino acid), position 60 (hydrophobic amino acid, particularly phenylalanine or tyrosine).
  • the disclosure provides a framework of amino acids that may be conserved.
  • Other positions that may be desirable to conserve are as follows: position 52 (acidic amino acid), position 55 (basic amino acid), position 81 (acidic), 98 (polar or charged, particularly E, D, R or K), all with respect to SEQ ID NO:1.
  • ActRIIA polypeptide is one that comprises a polypeptide that starts at amino acid 30 and ends at amino acid 110 of SEQ ID NO:9. Accordingly, ActRIIA polypeptides and ActRIIA-based GDF traps of the present disclosure may comprise a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 30-110 of SEQ ID NO:9. In some embodiments, ActRIIA-based GDF traps of the present disclosure do not comprise or consist of amino acids 30-110 of SEQ ID NO:9.
  • ActRIIA polypeptides and ActRIIA-based GDF trap polypeptides of the present disclosure comprise a polypeptide that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids amino acids 12-82 of SEQ ID NO:9 optionally beginning at a position ranging from 1-5 ( e.g., 1, 2, 3, 4, or 5) or 3-5 ( e.g., 3, 4, or 5) and ending at a position ranging from 110-116 ( e.g., 110, 111, 112, 113, 114, 115, or 116) or 110-115 ( e.g., 110, 111, 112, 113, 114, or 115), respectively, and comprising no more than 1, 2, 5, 10 or 15 conservative amino acid changes in the ligand binding pocket, and zero, one or more non-conservative alterations at positions 40, 53, 55, 74, 79 and/or 82 in the ligand-binding pocket with respect to
  • functionally active fragments of ActRII polypeptides e.g . ActRIIA and ActRIIB polypeptides
  • GDF trap polypeptides of the present disclosure can be obtained by screening polypeptides recombinantly produced from the corresponding fragment of the nucleic acid encoding an ActRII polypeptide or GDF trap polypeptide (e.g ., SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 46, and 48).
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments that can function as antagonists (inhibitors) of ActRII receptors and/or one or more ActRII ligands (e.g ., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and/or Nodal).
  • ActRII ligands e.g ., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and/or Nodal.
  • an ActRIIA polypeptide of the present disclosure is a polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, and 28.
  • the ActRIIA polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, and 28.
  • the ActRIIA polypeptide consists essentially of, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, and 28.
  • an ActRIIB polypeptide of the present disclosure is a polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 31, and 49.
  • the ActRIIB polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 31, and 49.
  • the ActRIIB polypeptide consists essentially of, or consists of, an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 31, and 49.
  • a GDF trap polypeptide of the present disclosure is a variant ActRIIB polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31, 36, 37, 38, 41, 44, 45, 49, 50, and 51.
  • the GDF trap comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31, 36, 37, 38, 41, 44, 45, 49, 50, and 51.
  • the GDF trap comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, 30, 31, 36, 37, 38, 41, 44, 45, 49, 50, and 51, wherein the position corresponding to L79 of SEQ ID NO:1, 4, or 49 is an acidic amino acids (a D or E amino acid residue).
  • the GDF trap consists essentially of, or consists of, an amino acid sequence that at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 36, 37, 38, 41, 44, 45, 50, and 51. In certain embodiments, the GDF trap does not comprise or consists of an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 29, and 31.
  • a GDF trap polypeptide of the present disclosure is a variant ActRIIA polypeptide comprising an amino acid sequence that is at least 75% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31.
  • the GDF trap comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31.
  • the GDF trap consists essentially of, or consists of, an amino acid sequence that at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31. In certain embodiments, the GDF trap does not comprise or consists of an amino acid sequence selected from SEQ ID NOs: 9, 10, 11, 22, 26, 28, 29, and 31.
  • the present disclosure contemplates making functional variants by modifying the structure of an ActRII polypeptide (e.g . and ActRIIA or ActRIIB polypeptide) or a GDF trap for such purposes as enhancing therapeutic efficacy, or stability (e.g., shelf-life and resistance to proteolytic degradation in vivo ) .
  • Variants can be produced by amino acid substitution, deletion, addition, or combinations thereof.
  • Whether a change in the amino acid sequence of a polypeptide of the disclosure results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in cells in a fashion similar to the wild-type polypeptide, or to bind to one or more ligands, such as GDF11, activin A, activin B, activin AB, activin C, activin E, GDF8, BMP6, and BMP7, as compared to the unmodified or a wild-type polypeptide.
  • ligands such as GDF11, activin A, activin B, activin AB, activin C, activin E, GDF8, BMP6, and BMP7
  • the present disclosure contemplates specific mutations of ActRII polypeptides and GDF trap polypeptides of the present disclosure so as to alter the glycosylation of the polypeptide.
  • Such mutations may be selected so as to introduce or eliminate one or more glycosylation sites, such as O-linked or N-linked glycosylation sites.
  • Asparagine-linked glycosylation recognition sites generally comprise a tripeptide sequence, asparagine-X-threonine or asparagine-X-serine (where "X" is any amino acid) which is specifically recognized by appropriate cellular glycosylation enzymes.
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the polypeptide (for O-linked glycosylation sites).
  • a variety of amino acid substitutions or deletions at one or both of the first or third amino acid positions of a glycosylation recognition site (and/or amino acid deletion at the second position) results in non-glycosylation at the modified tripeptide sequence.
  • Another means of increasing the number of carbohydrate moieties on a polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide.
  • the sugar(s) may be attached to (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those of cysteine; (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan; or (f) the amide group of glutamine. Removal of one or more carbohydrate moieties present on a polypeptide may be accomplished chemically and/or enzymatically.
  • Chemical deglycosylation may involve, for example, exposure of a polypeptide to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the amino acid sequence intact.
  • Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. [Meth. Enzymol. (1987) 138:350 ].
  • the sequence of a polypeptide may be adjusted, as appropriate, depending on the type of expression system used, as mammalian, yeast, insect, and plant cells may all introduce differing glycosylation patterns that can be affected by the amino acid sequence of the peptide.
  • ActRII polypeptides and GDF trap polypeptides of the present disclosure for use in humans may be expressed in a mammalian cell line that provides proper glycosylation, such as HEK293 or CHO cell lines, although other mammalian expression cell lines are expected to be useful as well.
  • This disclosure further contemplates a method of generating mutants, particularly sets of combinatorial mutants of ActRII polypeptides and GDF trap polypeptides of the present disclosure, as well as truncation mutants. Pools of combinatorial mutants are especially useful for identifying ActRII and GDF trap sequences.
  • the purpose of screening such combinatorial libraries may be to generate, for example, polypeptides variants which have altered properties, such as altered pharmacokinetic or altered ligand binding.
  • a variety of screening assays are provided below, and such assays may be used to evaluate variants.
  • ActRII polypeptides and GDF trap polypeptides may be screened for ability to bind to an ActRII receptor, to prevent binding of an ActRII ligand (e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal) to an ActRII polypeptide, or to interfere with signaling caused by an ActRII ligand.
  • an ActRII ligand e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal
  • the activity of an ActRII polypeptides or GDF trap polypeptides may also be tested in a cell-based or in vivo assay.
  • the effect of an ActRII polypeptide or GDF trap polypeptide on the expression of genes involved in hematopoiesis may be assessed. This may, as needed, be performed in the presence of one or more recombinant ActRII ligand proteins (e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal), and cells may be transfected so as to produce an ActRII polypeptide or GDF trap polypeptide, and optionally, an ActRII ligand.
  • ActRII ligand proteins e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal
  • an ActRII polypeptide or GDF trap polypeptide may be administered to a mouse or other animal, and one or more blood measurements, such as an RBC count, hemoglobin, or reticulocyte count may be assessed using art-recognized methods.
  • Combinatorial-derived variants can be generated which have a selective or generally increased potency relative to a reference ActRII polypeptide or GDF trap polypeptide.
  • Such variants when expressed from recombinant DNA constructs, can be used in gene therapy protocols.
  • mutagenesis can give rise to variants which have intracellular half-lives dramatically different than the corresponding unmodified ActRII polypeptide or GDF trap polypeptide.
  • the altered protein can be rendered either more stable or less stable to proteolytic degradation or other cellular processes which result in destruction,or otherwise inactivation, of an unmodified polypeptide.
  • Such variants can be utilized to alter ActRII polypeptide or GDF trap polypeptide levels by modulating the half-life of the polypeptide. For instance, a short half-life can give rise to more transient biological effects and, when part of an inducible expression system, can allow tighter control of recombinant ActRII polypeptide or GDF trap polypeptide levels within the cell.
  • mutations may be made in the linker (if any) and/or the Fc portion to alter the half-life of the protein.
  • a combinatorial library may be produced by way of a degenerate library of genes encoding a library of polypeptides which each include at least a portion of potential ActRII or GDF trap sequences.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential ActRII or GDF trap polypeptide encoding nucleotide sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins ( e.g ., for phage display).
  • the library of potential homologs can be generated from a degenerate oligonucleotide sequence.
  • Chemical synthesis of a degenerate gene sequence can be carried out in an automatic DNA synthesizer, and the synthetic genes can then be ligated into an appropriate vector for expression.
  • the synthesis of degenerate oligonucleotides is well known in the art. See, e.g ., Narang, SA (1983) Tetrahedron 39:3 ; Itakura et al. (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289 ; Itakura et al.
  • ActRII polypeptides or GDF trap polypeptides of the present disclosure can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis [see, e.g., Ruf et al. (1994) Biochemistry 33:1565-1572 ; Wang et al. (1994) J. Biol. Chem. 269:3095-3099 ; Balint et al. (1993) Gene 137:109-118 ; Grodberg et al. (1993) Eur. J. Biochem. 218:597-601 ; Nagashima et al. (1993) J. Biol. Chem.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations and truncations, and, for that matter, for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of ActRII polypeptides or GDF trap polypeptides of the disclosure.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Preferred assays include ActRII ligand (e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal) binding assays and/or ActRII ligand-mediated cell signaling assays.
  • ActRII ligand e.g., GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP7, BMP6, and/or Nodal binding assays and/or ActRII ligand-mediated cell signaling assays.
  • ActRII polypeptides or GDF trap polypeptides of the present disclosure may further comprise post-translational modifications in addition to any that are naturally present in the ActRII (e.g., an ActRIIA or ActRIIB polypeptide) or GDF trap polypeptide.
  • Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • the ActRII polypeptide or GDF trap polypeptide may contain non-amino acid elements, such as polyethylene glycols, lipids, polysaccharide or monosaccharide, and phosphates.
  • ActRII polypeptides or GDF trap polypeptides of the present disclosure include fusion proteins having at least a portion (domain) of an ActRII polypeptide (e.g., an ActRIIA or ActRIIB polypeptide) or GDF trap polypeptide and one or more heterologous portions (domains).
  • fusion domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy-chain constant region (Fc), maltose binding protein (MBP), or human serum albumin.
  • a fusion domain may be selected so as to confer a desired property.
  • fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography.
  • relevant matrices for affinity chromatography such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used.
  • Many of such matrices are available in "kit” form, such as the Pharmacia GST purification system and the QIAexpressTM system (Qiagen) useful with (HIS 6 ) fusion partners.
  • a fusion domain may be selected so as to facilitate detection of the ligand trap polypeptides.
  • detection domains include the various fluorescent proteins (e.g ., GFP) as well as "epitope tags," which are usually short peptide sequences for which a specific antibody is available.
  • epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags.
  • the fusion domains have a protease cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.
  • an ActRII polypeptide or a GDF trap polypeptide is fused with a domain that stabilizes the polypeptide in vivo (a "stabilizer” domain).
  • stabilizing is meant anything that increases serum half-life, regardless of whether this is because of decreased destruction, decreased clearance by the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an immunoglobulin are known to confer desirable pharmacokinetic properties on a wide range of proteins. Likewise, fusions to human serum albumin can confer desirable properties.
  • Other types of fusion domains that may be selected include multimerizing (e.g ., dimerizing, tetramerizing) domains and functional domains (that confer an additional biological function, such as further stimulation of muscle growth).
  • the present disclosure provides ActRII or GDF trap fusion proteins comprising the following IgG1 Fc domain sequence:
  • the present disclosure provides ActRII or GDF trap fusion proteins comprising the following variant of the IgG1 Fc domain:
  • the present disclosure provides ActRII or GDF trap fusion proteins comprising the following variant of the IgG1 Fc domain:
  • the IgG1 Fc domain has one or more mutations at residues such as Asp-265, lysine 322, and Asn-434.
  • the mutant IgG1 Fc domain having one or more of these mutations e.g., Asp-265 mutation
  • the mutant Fc domain having one or more of these mutations has increased ability of binding to the MHC class I-related Fc-receptor (FcRN) relative to a wild-type IgG1 Fc domain.
  • the present disclosure provides ActRII or GDF trap fusion proteins comprising variants of the IgG2 Fc domain, including the following:
  • an ActRII polypeptide domain or GDF trap polypeptide domain may be placed C-terminal to a heterologous domain, or alternatively, a heterologous domain may be placed C-terminal to an ActRII polypeptide domain or GDF trap polypeptide domain.
  • the ActRII polypeptide domain or GDF trap polypeptide domain and the heterologous domain need not be adjacent in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains.
  • an ActRII or GDF trap fusion protein may comprise an amino acid sequence as set forth in the formula A-B-C.
  • the B portion corresponds to an ActRII polypeptide domain or a GDF trap polypeptide domain.
  • the A and C portions may be independently zero, one, or more than one amino acid, and both the A and C portions when present are heterologous to B.
  • the A and/or C portions may be attached to the B portion via a linker sequence.
  • Exemplary linkers include short polypeptide linkers such as 2-10, 2-5, 2-4, 2-3 glycine residues, such as, for example, a Gly-Gly-Gly linker.
  • an ActRII or GDF trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a leader (signal) sequence, B consists of an ActRII or GDF polypeptide domain, and C is a polypeptide portion that enhances one or more of in vivo stability, in vivo half-life, uptake/administration, tissue localization or distribution, formation of protein complexes, and/or purification.
  • an ActRII or GDF trap fusion protein comprises an amino acid sequence as set forth in the formula A-B-C, wherein A is a TPA leader sequence, B consists of an ActRII or GDF polypeptide domain, and C is an immunoglobulin Fc domain.
  • Preferred fusion proteins comprises the amino acid sequence set forth in any one of SEQ ID NOs: 22, 26, 29, 31, 36, 38, 41, 44, and 51.
  • ActRII polypeptides or GDF trap polypeptides of the present disclosure contain one or more modifications that are capable of stabilizing the polypeptides.
  • modifications enhance the in vitro half-life of the polypeptides, enhance circulatory half-life of the polypeptides, and/or reduce proteolytic degradation of the polypeptides.
  • Such stabilizing modifications include, but are not limited to, fusion proteins (including, for example, fusion proteins comprising an ActRII polypeptide domain or a GDF trap polypeptide domain and a stabilizer domain), modifications of a glycosylation site (including, for example, addition of a glycosylation site to a polypeptide of the disclosure), and modifications of carbohydrate moiety (including, for example, removal of carbohydrate moieties from a polypeptide of the disclosure).
  • fusion proteins including, for example, fusion proteins comprising an ActRII polypeptide domain or a GDF trap polypeptide domain and a stabilizer domain
  • modifications of a glycosylation site including, for example, addition of a glycosylation site to a polypeptide of the disclosure
  • modifications of carbohydrate moiety including, for example, removal of carbohydrate moieties from a polypeptide of the disclosure.
  • stabilizer domain not only refers to a fusion domain (e.g ., an immunoglobulin Fc domain) as in the case of fusion proteins, but also includes nonproteinaceous modifications such as a carbohydrate moiety, or nonproteinaceous moiety, such as polyethylene glycol.
  • ActRII polypeptides and GDF traps to be used in accordance with the methods described herein are isolated polypeptides.
  • an isolated protein or polypeptide is one which has been separated from a component of its natural environment.
  • a polypeptide of the disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity as determined by, for example, electrophoretic (e.g ., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g ., ion exchange or reverse phase HPLC).
  • electrophoretic e.g ., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g ., ion exchange or reverse phase HPLC
  • ActRII polypeptides and GDF traps of the disclosure can be produced by a variety of art-known techniques.
  • polypeptides of the disclosure can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993 ) and Grant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992 ).
  • automated peptide synthesizers are commercially available (see, e.g., Advanced ChemTech Model 396; Milligen/Biosearch 9600).
  • polypeptides of the disclosure may be recombinantly produced using various expression systems [ e.g ., E. coli, Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus] as is well known in the art.
  • the modified or unmodified polypeptides of the disclosure may be produced by digestion of recombinantly produced full-length ActRII or GDF trap polypeptides by using, for example, a protease, e.g ., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE).
  • a protease e.g ., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE).
  • PACE paired basic amino acid converting enzyme
  • Computer analysis using a commercially available software, e.g ., MacVector, Omega, PCGene, Molecular Simulation, Inc. can be used to identify proteolytic cleavage sites.
  • polypeptides may be produced from recombinantly produced full-length ActRII or GDF trap polypeptides using chemical cleavage ( e.g ., cyanogen bromide, hydroxylamine, etc. ) .
  • ActRII polypeptides disclosed herein can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g ., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • additional ActRII antagonist agents of the disclosure e.g ., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • an ActRII polypeptide disclosed herein can be used in combination with i) one or more additional ActRII polypeptides disclosed herein, ii) one or more GDF traps disclosed herein; iii) one or more ActRII antagonist antibodies disclosed herein (e.g., an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIB antibody); iv) one or more small-molecule ActRII antagonists disclosed herein (e.g., a small-molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB); v) one or
  • any of the GDF traps disclosed herein can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a patient in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • a GDF trap disclosed herein can be used in combination with i) one or more additional GDF traps disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g ., ActRIIA or ActRIIB polypeptides) disclosed herein; iii) one or more ActRII antagonist antibodies disclosed herein (e.g., an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIB antibody); iv) one or more small-molecule ActRII antagonists disclosed herein (e.g., a small-molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7
  • the present disclosure provides isolated and/or recombinant nucleic acids encoding the ActRII polypeptides and GDF trap polypeptides (including fragments, functional variants, and fusion proteins thereof) disclosed herein.
  • SEQ ID NO:12 encodes the naturally occurring human ActRIIA precursor polypeptide
  • SEQ ID NO:13 encodes the processed extracellular domain of ActRIIA.
  • SEQ ID NO:7 encodes a naturally occurring human ActRIIB precursor polypeptide (the R64 variant described above)
  • SEQ ID NO:8 encodes the processed extracellular domain of ActRIIB (the R64 variant described above).
  • the subject nucleic acids may be single-stranded or double stranded. Such nucleic acids may be DNA or RNA molecules. These nucleic acids may be used, for example, in methods for making ActRII-based ligand trap polypeptides of the present disclosure.
  • isolated nucleic acid(s) refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • nucleic acids encoding ActRII polypeptides and GDF traps of the present disclosure are understood to include nucleic acids that are variants of any one of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48.
  • Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions, or deletions including allelic variants, and therefore, will include coding sequence that differ from the nucleotide sequence designated in any one of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48.
  • ActRII polypeptides and GDF traps of the present disclosure are encoded by isolated or recombinant nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48
  • GDF traps of the present disclosure are not encoded by nucleic acid sequences that comprise or consist of any one of nucleotide sequences corresponding to any one of SEQ ID NOs: 7, 8, 12, 13, 27, and 32.
  • nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the sequences complementary to SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 42, 47, and 48, and variants thereof, are also within the scope of the present disclosure.
  • the nucleic acid sequences of the disclosure can be isolated, recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA library.
  • nucleic acids of the present disclosure also include nucleotide sequences that hybridize under highly stringent conditions to the nucleotide sequence designated in SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48, complement sequences of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48, or fragments thereof.
  • SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48 complement sequences of SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48, or fragments thereof.
  • the hybridization at 6.0 x sodium chloride/sodium citrate (SSC) at about 45 °C, followed by a wash of 2.0 x SSC at 50 °C.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50 °C to a high stringency of about 0.2 x SSC at 50 °C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22 °C, to high stringency conditions at about 65 °C. Both temperature and salt may be varied, or temperature or salt concentration may be held constant while the other variable is changed.
  • the disclosure provides nucleic acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature.
  • Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ ID NOs: 7, 8, 12, 13, 27, 32, 39, 40, 42, 43, 46, 47, and 48 due to degeneracy in the genetic code are also within the scope of the disclosure.
  • a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in "silent" mutations which do not affect the amino acid sequence of the protein.
  • CAU and CAC are synonyms for histidine
  • nucleotides up to about 3-5% of the nucleotides
  • nucleic acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this disclosure.
  • the recombinant nucleic acids of the present disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct.
  • Regulatory nucleotide sequences will generally be appropriate to the host cell used for expression.
  • suitable regulatory sequences are known in the art for a variety of host cells.
  • said one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the disclosure.
  • the promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.
  • An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.
  • the subject nucleic acid is provided in an expression vector comprising a nucleotide sequence encoding an ActRII polypeptide or a GDF trap and operably linked to at least one regulatory sequence.
  • Regulatory sequences are art-recognized and are selected to direct expression of the ActRII or GDF trap polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, CA (1990 ).
  • any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding an ActRII or GDF trap polypeptide.
  • useful expression control sequences include, for example, the early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus immediate early promoter, RSV promoters, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g ., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression
  • the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other protein encoded by the vector, such as antibiotic markers, should also be considered.
  • a recombinant nucleic acid of the present disclosure can be produced by ligating the cloned gene, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells (yeast, avian, insect or mammalian), or both.
  • Expression vehicles for production of a recombinant ActRII or GDF trap polypeptide include plasmids and other vectors.
  • suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • Some mammalian expression vectors contain both prokaryotic sequences to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • bacterial plasmids such as pBR322
  • derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papilloma virus
  • pHEBo Epstein-Barr virus
  • pREP-derived and p205 Epstein-Barr virus
  • examples of other viral (including retroviral) expression systems can be found below in the description of gene therapy delivery systems.
  • the various methods employed in the preparation of the plasmids and in transformation of host organisms are well known in the art.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as the ⁇ -gal containing pBlueBac III).
  • pVL-derived vectors such as pVL1392, pVL1393 and pVL941
  • pAcUW-derived vectors such as pAcUW1
  • pBlueBac-derived vectors such as the ⁇ -gal containing pBlueBac III.
  • a vector will be designed for production of the subject ActRII or GDF trap polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wise.).
  • a vector will be designed for production of the subject ActRII or GDF trap polypeptides in CHO cells, such as a Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wise.).
  • the subject gene constructs can be used to cause expression of the subject ActRII polypeptides in cells propagated in culture, e.g., to produce proteins, including fusion proteins or variant proteins, for purification.
  • This disclosure also pertains to a host cell transfected with a recombinant gene including a coding sequence for one or more of the subject ActRII or GDF trap polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • an ActRII or GDF trap polypeptide of the disclosure may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast, or mammalian cells [ e.g. a Chinese hamster ovary (CHO) cell line].
  • Other suitable host cells are known to those skilled in the art.
  • the present disclosure further pertains to methods of producing the subject ActRII and GDF trap polypeptides.
  • a host cell transfected with an expression vector encoding an ActRII or GDF trap polypeptide can be cultured under appropriate conditions to allow expression of the ActRII or GDF trap polypeptide to occur.
  • the polypeptide may be secreted and isolated from a mixture of cells and medium containing the polypeptide.
  • the ActRII or GDF trap polypeptide may be retained cytoplasmically or in a membrane fraction and the cells harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • the subject polypeptides can be isolated from cell culture medium, host cells, or both, using techniques known in the art for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, immunoaffinity purification with antibodies specific for particular epitopes of the ActRII or GDF trap polypeptides, and affinity purification with an agent that binds to a domain fused to the ActRII or GDF trap polypeptide (e.g., a protein A column may be used to purify an ActRII-Fc or GDF Trap-Fc fusion protein).
  • the ActRII or GDF trap polypeptide is a fusion protein containing a domain which facilitates its purification.
  • purification is achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • An ActRII-Fc or GDF trap-Fc protein may be purified to a purity of >90%, >95%, >96%, >98%, or >99% as determined by size exclusion chromatography and >90%, >95%, >96%, >98%, or >99% as determined by SDS PAGE.
  • the target level of purity should be one that is sufficient to achieve desirable results in mammalian systems, particularly non-human primates, rodents (mice), and humans.
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant ActRII or GDF trap polypeptide, can allow purification of the expressed fusion protein by affinity chromatography using a Ni 2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified ActRII or GDF trap polypeptide. See, e.g., Hochuli et al. (1987) J. Chromatography 411:177 ; and Janknecht et al. (1991) PNAS USA 88:8972 .
  • fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence. See, e.g ., Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992 .
  • the present disclosure relates to an antibody, or combination of antibodies, that antagonize ActRII activity (e.g ., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling).
  • ActRII activity e.g ., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling.
  • Such antibodies may bind to and inhibit one or more TGF- ⁇ family ligands (e.g.
  • the disclosure provides methods of using an antibody ActRII antagonist or combination of antibody ActRII antagonists, alone or in combination with one or more erythropoiesis stimulating agents (e.g ., EPO) or other supportive therapies [ e.g ., transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea, etc.
  • EPO erythropoiesis stimulating agents
  • supportive therapies e.g ., transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea, etc.
  • sickle-cell disease in a subject in need thereof, treat or prevent one or more complication of sickle-cell disease (e.g ., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis, aneurysm, ischemic stroke, intraparenchymal hemorrhage, subarachnoid hemorrh
  • a preferred antibody ActRII antagonist of the disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of at least GDF11 (e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • GDF11 e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • the antibody, or combination of antibodies further binds to and/or inhibits activity of GDF8 (e.g ., GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly in the case of a multispecific antibody that has binding affinity for both GDF11 and GDF8 or in the context of a combination of one or more anti-GDFl 1 antibodies and one or more anti-GDF8 antibodies.
  • an antibody, or combination of antibodies, of the disclosure does not substantially bind to and/or inhibit activity of activin A (e.g ., activin A-mediated activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • an antibody, or combination of antibodies, of the disclosure that binds to and/or inhibits the activity of GDF11 and/or GDF8 further binds to and/or inhibits activity of one of more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal (e.g ., activation of ActRIIA or ActRIIB SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the case of a multispecific antibody that has binding affinity for multiple ActRII ligands or in the context of a combination of multiple antibodies - each having binding affinity for a different ActRII ligand.
  • an ActRII antagonist of the present disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of at least GDF8 (e.g ., GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • GDF8 e.g ., GDF8-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • the antibody, or combination of antibodies further binds to and/or inhibits activity of GDF11 (e.g., GDF11-mediated activation of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling), particularly in the case of a multispecific antibody that has binding affinity for both GDF8 and GDF11 or in the context of a combination of one or more anti-GDF8 antibodies and one or more anti-GDFl 1 antibodies.
  • an antibody, or combination of antibodies, of the disclosure does not substantially bind to and/or inhibit activity of activin A (e.g ., activin A-mediated activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • an antibody, or combination of antibodies, of the disclosure that binds to and/or inhibits the activity of GDF8 and/or GDF11 further binds to and/or inhibits activity of one of more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal (e.g. , activation of ActRIIA or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling), particularly in the case of a multispecific antibody that has binding affinity for multiple ActRII ligands or in the context of a combination multiple antibodies - each having binding affinity for a different ActRII ligand.
  • an ActRII antagonist of the present disclosure is an antibody, or combination of antibodies, that binds to and/or inhibits activity of an ActRII receptor (e.g. an ActRIIA or ActRIIB receptor).
  • an anti-ActRII receptor antibody e.g. an anti-ActRIIA or anti-ActRIIB receptor antibody
  • an anti-ActRII receptor antibody, or combination of antibodies, of the disclosure further prevents binding and/or activation of the ActRII receptor by GDF8.
  • an anti-ActRII receptor antibody, or combination of antibodies, of the disclosure does not substantially inhibit activin A from binding to and/or activating an ActRII receptor.
  • an anti-ActRII receptor antibody, or combination of antibodies, of the disclosure that binds to an ActRII receptor and prevents binding and/or activation of the ActRII receptor by GDF11 and/or GDF8 further prevents binding and/or activation of the ActRII receptor by one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies ( e.g ., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.
  • An antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules ( e.g ., scFv); and multispecific antibodies formed from antibody fragments.
  • Antibodies disclosed herein may be polyclonal antibodies or monoclonal antibodies.
  • the antibodies of the present disclosure comprise a label attached thereto and able to be detected (e.g., the label can be a radioisotope, fluorescent compound, enzyme, or enzyme co-factor).
  • the antibodies of the present disclosure are isolated antibodies.
  • Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097 ; WO 1993/01161 ; Hudson et al. (2003) Nat. Med. 9:129-134 (2003 ); and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448 . Triabodies and tetrabodies are also described in Hudson et al. (2003) Nat. Med. 9:129-134 .
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy-chain variable domain or all or a portion of the light-chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody. See, e.g., U.S. Pat. No. 6,248,516 .
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.
  • recombinant host cells e.g., E. coli or phage
  • the antibodies herein may be of any class.
  • the class of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu.
  • an antibody for use in the methods disclosed herein specifically binds to its target antigen, preferably with high binding affinity.
  • Affinity may be expressed as a K D value and reflects the intrinsic binding affinity (e.g ., with minimized avidity effects).
  • binding affinity is measured in vitro, whether in a cell-free or cell-associated setting. Any of a number of assays known in the art, including those disclosed herein, can be used to obtain binding affinity measurements including, for example, surface plasmon resonance (BiacoreTM assay), radiolabeled antigen binding assay (RIA), and ELISA.
  • antibodies of the present disclosure bind to their target antigens (e.g .
  • GDF11, GDF8, ActRIIA, ActRIIB, etc. with at least a K D of 1x 10 -7 or stronger, 1x10 -8 or stronger, 1x10 -9 or stronger, 1x10 -10 or stronger, 1x10 -11 or stronger, 1x10 -12 or stronger, 1x10 -13 or stronger, or 1x10 -14 or stronger.
  • K D is measured by RIA performed with the Fab version of an antibody of interest and its target antigen as described by the following assay.
  • Solution binding affinity of Fabs for the antigen is measured by equilibrating Fab with a minimal concentration of radiolabeled antigen (e.g ., 125 I-labeled) in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate [see, e.g., Chen et al. (1999) J. Mol. Biol. 293:865-881 ].
  • multi-well plates e.g., MICROTITER® from Thermo Scientific
  • a capturing anti-Fab antibody e.g ., from Cappel Labs
  • bovine serum albumin preferably at room temperature (approximately 23°C).
  • radiolabeled antigen are mixed with serial dilutions of a Fab of interest [e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599 ].
  • the Fab of interest is then incubated, preferably overnight but the incubation may continue for a longer period (e.g ., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation, preferably at room temperature for about one hour. The solution is then removed and the plate is washed times several times, preferably with polysorbate 20 and PBS mixture. When the plates have dried, scintillant (e.g ., MICROSCINT® from Packard) is added, and the plates are counted on a gamma counter (e.g ., TOPCOUNT® from Packard).
  • a gamma counter e.g ., TOPCOUNT® from Packard
  • K D is measured using surface plasmon resonance assays using, for example a BIACORE® 2000 or a BIACORE® 3000 (Biacore, Inc., Piscataway, N.J.) with immobilized antigen CM5 chips at about 10 response units (RU).
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • an antigen can be diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml (about 0.2 ⁇ M) before injection at a flow rate of 5 ⁇ l/minute to achieve approximately 10 response units (RU) of coupled protein.
  • 1 M ethanolamine is injected to block unreacted groups.
  • two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20®) surfactant (PBST) at at a flow rate of approximately 25 ⁇ l/min.
  • association rates (k on ) and dissociation rates (k off ) are calculated using, for example, a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (K D ) is calculated as the ratio k off / k on [see, e.g., Chen et al., (1999) J. Mol. Biol. 293:865-881 ].
  • anti-GDFl 1 antibody generally refers to an antibody that is capable of binding to GDF11 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF11.
  • the extent of binding of an anti-GDFl 1 antibody to an unrelated, non-GDFl 1 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to GDF11 as measured, for example, by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an anti-GDF11 antibody binds to an epitope of GDF11 that is conserved among GDF11 from different species.
  • an anti-GDFl 1 antibody of the present disclosure is an antagonist antibody that can inhibit GDF11 activity.
  • an anti-GDF11 antibody of the disclosure may inhibit GDF11 from binding to a cognate receptor (e.g., ActRIIA or ActRIIB receptor) and/or inhibit GDF11-mediated signal transduction (activation) of a cognate receptor, such as SMAD2/3 signaling by ActRIIA and/or ActRIIB receptors.
  • anti-GDFl 1 antibodies of the present disclosure do not substantially bind to and/or inhibit activity of activin A.
  • GDF11 has high sequence homology to GDF8 and therefore antibodies that bind and/or to GDF11, in some cases, may also bind to and/or inhibit GDF8.
  • an anti-GDF8 antibody refers to an antibody that is capable of binding to GDF8 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting GDF8.
  • the extent of binding of an anti-GDF8 antibody to an unrelated, non-GDF8 protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to GDF8 as measured, for example, by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an anti-GDF8 antibody binds to an epitope of GDF8 that is conserved among GDF8 from different species.
  • an anti-GDF8 antibody of the present disclosure is an antagonist antibody that can inhibit GDF8 activity.
  • an anti-GDF8 antibody of the disclosure may inhibit GDF8 from binding to a cognate receptor (e.g ., ActRIIA or ActRIIB receptor) and/or inhibit GDF8-mediated signal transduction (activation) of a cognate receptor, such as SMAD2/3 signaling by ActRIIA and/or ActRIIB receptors.
  • anti-GDF8 antibodies of the present disclosure do not substantially bind to and/or inhibit activity of activin A. It should be noted that GDF8 has high sequence homology to GDF11 and therefore antibodies that bind and/or to GDF8, in many cases, may also bind to and/or inhibit GDF11.
  • an anti-ActRIIA antibody refers to an antibody that is capable of binding to ActRIIA with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIA.
  • the extent of binding of an anti-ActRIIA antibody to an unrelated, non-ActRIIA protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to ActRIIA as measured, for example, by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an anti-ActRIIA antibody binds to an epitope of ActRIIA that is conserved among ActRIIA from different species.
  • an anti-ActRIIA antibody of the present disclosure is an antagonist antibody that can inhibit ActRIIA activity.
  • an anti-ActRIIA antibody of the present disclosure may inhibit one or more ActRIIA ligands selected from activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, activin A, BMP6, and BMP7 from binding to the ActRIIA receptor and/or inhibit one of these ligands from activating ActRIIA signaling (e.g ., SMAD2/3 and/or SMAD 1/5/8 ActRIIA signaling).
  • ActRIIA signaling e.g ., SMAD2/3 and/or SMAD 1/5/8 ActRIIA signaling.
  • anti-ActRIIA antibodies of the present disclosure inhibit GDF11 from binding to the ActRIIA receptor and/or inhibit GDF11 from activating ActRIIA signaling.
  • anti-ActRIIA antibodies of the disclosure further inhibit GDF8 from binding to the ActRIIA receptor and/or inhibit GDF8 from activating ActRIIA signaling.
  • anti-ActRIIA antibodies of the present disclosure do not substantially inhibit activin A from binding to the ActRIIA receptor and/or do not substantially inhibit activin A-mediated activation of ActRIIA signaling.
  • an anti-ActRIIA antibody of the disclosure that inhibits GDF11 and/or GDF8 from binding to and/or activating an ActRIIA receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, activin A, GDF8, BMP6, and BMP7 from binding to and/or activating the ActRIIA receptor.
  • an anti-ActRIIB antibody refers to an antibody that is capable of binding to ActRIIB with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting ActRIIB.
  • the extent of binding of an anti-ActRIIB antibody to an unrelated, non-ActRIIB protein is less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% of the binding of the antibody to ActRIIB as measured, for example, by a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • an anti-ActRIIB antibody binds to an epitope of ActRIIB that is conserved among ActRIIB from different species.
  • an anti-ActRIIB antibody of the present disclosure is an antagonist antibody that can inhibit ActRIIB activity.
  • an anti-ActRIIB antibody of the present disclosure may inhibit one or more ActRIIB ligands selected from activin A, activin B, activin AB, activin C, activin E, GDF11, GDF8, activin A, BMP6, and BMP7 from binding to the ActRIIB receptor and/or inhibit one of these ligands from activating ActRIIB signaling (e.g ., SMAD2/3 and/or SMAD 1/5/8 ActRIIB signaling).
  • ActRIIB signaling e.g ., SMAD2/3 and/or SMAD 1/5/8 ActRIIB signaling.
  • anti-ActRIIB antibodies of the present disclosure inhibit GDF11 from binding to the ActRIIB receptor and/or inhibit GDF11 from activating ActRIIB signaling.
  • anti-ActRIIB antibodies of the disclosure further inhibit GDF8 from binding to the ActRIIB receptor and/or inhibit GDF8 from activating ActRIIB signaling.
  • anti-ActRIIB antibodies of the present disclosure do not substantially inhibit activin A from binding to the ActRIIB receptor and/or do not substantially inhibit activin A-mediated activation of ActRIIB signaling.
  • an anti-ActRIIB antibody of the disclosure that inhibits GDF11 and/or GDF8 from binding to and/or activating an ActRIIB receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, activin A, GDF8, BMP6, and BMP7 from binding to and/or activating the ActRIIB receptor.
  • nucleic acid and amino acid sequences of human GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, GDF8, BMP6, BMP7, ActRIIB, and ActRIIA are well known in the art and thus antibody antagonists for use in accordance with this disclosure may be routinely made by the skilled artisan based on the knowledge in the art and teachings provided herein.
  • an antibody provided herein e.g., an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody
  • a chimeric antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567 ; and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855 .
  • a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region.
  • a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody.
  • chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody provided herein is a humanized antibody.
  • a humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human framework regions (FRs).
  • HVRs non-human hypervariable regions
  • FRs human framework regions
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g ., a non-human antibody refers to an antibody that has undergone humanization.
  • Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the "best-fit" method [see, e.g., Sims et al. (1993) J. Immunol. 151:2296 ]; framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light-chain or heavy-chain variable regions [see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285 ; and Presta et al. (1993) J. Immunol., 151:2623 ]; human mature (somatically mutated) framework regions or human germline framework regions [see, e.g ., Almagro and Fransson (2008) Front.
  • an antibody provided herein e.g., an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody
  • Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel (2001) Curr. Opin. Pharmacol. 5: 368-74 and Lonberg (2008) Curr. Opin. Immunol. 20:450-459 .
  • Human antibodies may be prepared by administering an immunogen (e.g ., a GDF11 polypeptide, GDF8 polypeptide, an ActRIIA polypeptide, or an ActRIIB polypeptide) to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • an immunogen e.g ., a GDF11 polypeptide, GDF8 polypeptide, an ActRIIA polypeptide, or an ActRIIB polypeptide
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated.
  • Human variable regions from intact antibodies generated by such animals may be further modified, for example, by combining with a different human constant region.
  • Human antibodies provided herein can also be made by hybridoma-based methods.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described [see, e.g., Kozbor J. Immunol., (1984) 133: 3001 ; Brodeur et al. (1987) Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York ; and Boerner et al. (1991) J. Immunol., 147: 86 ].
  • Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., (2006) Proc. Natl. Acad. Sci.
  • Human antibodies provided herein may also be generated by isolating Fv clone variable-domain sequences selected from human-derived phage display libraries. Such variable-domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described herein.
  • antibodies of the present disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities.
  • a variety of methods are known in the art for generating phage-display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, N.J . and further described, for example, in the McCafferty et al. (1991) Nature 348:552-554 ; Clackson et al., (1991) Nature 352: 624-628 ; Marks et al. (1992) J. Mol. Biol.
  • repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al. (1994) Ann. Rev. Immunol., 12: 433-455 .
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • Libraries from immunized sources provide high-affinity antibodies to the immunogen (e.g ., GDF11, activin B, ActRIIA, or ActRIIB) without the requirement of constructing hybridomas.
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies directed against a wide range of non-self and also self-antigens without any immunization as described by Griffiths et al. (1993) EMBO J, 12: 725-734 .
  • naive libraries can also be made synthetically by cloning un-rearranged V-gene segments from stem cells and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter (1992) J. Mol. Biol., 227: 381-388 .
  • Patent publications describing human antibody phage libraries include, for example: U.S. Pat. No.
  • an antibody provided herein is a multispecific antibody, for example, a bispecific antibody.
  • Multispecific antibodies typically monoclonal antibodies
  • a multispecific antibody of the present disclosure comprises two or more binding specificities, with at least one of the binding specificities being for a GDF11 epitope, and optionally one or more additional binding specificities being for an epitope on a different ActRII ligand (e.g ., GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6 BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor).
  • multispecific antibodies may bind to two or more different epitopes of GDF11.
  • a multispecific antibody of the disclosure that has binding affinity, in part, for a GDF11 epitope can be used to inhibit a GDF11 activity (e.g., the ability to bind to and/or activate an ActRIIA and/or ActRIIB receptor), and optionally inhibit the activity of one or more different ActRII ligands (e.g ., GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA or ActRIIB receptor).
  • a GDF11 activity e.g., the ability to bind to and/or activate an ActRIIA and/or ActRIIB receptor
  • an ActRII ligands e.g ., GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal
  • an ActRII receptor e.
  • multispecific antibodies of the present disclosure that bind to and/or inhibit GDF11 further bind to and/or inhibit at least GDF8.
  • multispecific antibodies of the disclosure that bind to and/or inhibit GDF11 do not substantially bind to and/or substantially inhibit activin A.
  • multispecific antibodies of the disclosure that bind to and/or inhibit GDF11 and GDD8 further bind to and/or inhibit one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal.
  • a multispecific antibody of the present disclosure comprises two or more binding specificities, with at least one of the binding specificities being for a GDF8 epitope, and optionally one or more additional binding specificities being for an epitope on a different ActRII ligand (e.g., GDF11, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA and/or ActRIIB receptor).
  • multispecific antibodies may bind to two or more different epitopes of GDF8.
  • a multispecific antibody of the disclosure that has binding affinity, in part, for an GDF8 epitope can be used to inhibit an GDF8 activity (e.g., the ability to bind to and/or activate an ActRIIA and/or ActRIIB receptor), and optionally inhibit the activity of one or more different ActRII ligands (e.g ., GDF11, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal) and/or an ActRII receptor (e.g., an ActRIIA or ActRIIB receptor).
  • an ActRII ligands e.g ., GDF11, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal
  • an ActRII receptor e.g., an ActRIIA or ActRIIB receptor
  • multispecific antibodies of the present disclosure that bind to and/or inhibit GDF8 further bind to and/or inhibit at least GDF11.
  • multispecific antibodies of the disclosure that bind to and/or inhibit GDF8 do not substantially bind to and/or substantially inhibit activin A.
  • multispecific antibodies of the disclosure that bind to and/or inhibit GDF8 and GDF11 further bind to and/or inhibit one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7 and/or Nodal.
  • the antibodies disclosed herein are monoclonal antibodies.
  • Monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g ., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present methods may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols [see, e.g ., Antibodies: A Laboratory Manual (1988) ed. by Harlow and Lane, Cold Spring Harbor Press ].
  • a mammal, such as a mouse, hamster, or rabbit can be immunized with an immunogenic form of the GDF11 or GDF8 polypeptide, an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of a GDF11 or GDF8 polypeptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibody production and/or level of binding affinity.
  • antibody-producing cells can be harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield hybridoma cells.
  • immortalizing cells such as myeloma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with a GDF11 or GDF8 polypeptide, and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein (e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody), thereby generating an Fc-region variant.
  • the Fc-region variant may comprise a human Fc-region sequence (e.g ., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution, deletion, and/or addition) at one or more amino acid positions.
  • the present disclosure contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet for which certain effector functions [ e.g., complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC)] are unnecessary or deleterious.
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities.
  • Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks Fc ⁇ R binding (hence likely lacking ADCC activity), but retains FcRn binding ability.
  • NK cells express Fc ⁇ RIII only, whereas monocytes express Fc ⁇ RI, Fc ⁇ RII and Fc ⁇ RIII.
  • FcR expression on hematopoietic cells is summarized in, for example, Ravetch and Kinet (1991) Annu. Rev. Immunol. 9:457-492 .
  • Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 ; Hellstrom, I. et al. (1986) Proc. Nat'l Acad. Sci. USA 83:7059-7063 ; Hellstrom, I et al. (1985) Proc. Nat'l Acad. Sci.
  • non-radioactive assay methods may be employed (e.g., ACTITM, non-radioactive cytotoxicity assay for flow cytometry; CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96® non-radioactive cytotoxicity assay, Promega, Madison, Wis.).
  • Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.
  • ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al. (1998) Proc. Nat'l Acad. Sci. USA 95:652-656 .
  • C1q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity [see, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402 ].
  • a CDC assay may be performed [see, e.g., Gazzano-Santoro et al. (1996) J. Immunol.
  • FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art [see, e.g., Petkova, S. B. et al. (2006) Int. Immunol. 18(12):1759-1769 ].
  • Antibodies of the present disclosure include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 ( U.S. Pat. No. 6,737,056 ).
  • Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine ( U.S. Pat. No. 7,332,581 ).
  • cysteine-engineered antibodies e.g., "thioMAbs”
  • one or more residues of an antibody are substituted with cysteine residues.
  • the substituted residues occur at accessible sites of the antibody.
  • reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein.
  • any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy-chain Fc region.
  • Cysteine engineered antibodies may be generated as described, for example., in U.S. Pat. No. 7,521,541 .
  • the techniques used to screen antibodies in order to identify a desirable antibody may influence the properties of the antibody obtained. For example, if an antibody is to be used for binding an antigen in solution, it may be desirable to test solution binding.
  • a variety of different techniques are available for testing interaction between antibodies and antigens to identify particularly desirable antibodies. Such techniques include ELISAs, surface plasmon resonance binding assays (e.g., the BiacoreTM binding assay, Biacore AB, Uppsala, Sweden), sandwich assays (e.g., the paramagnetic bead system of IGEN International, Inc., Gaithersburg, Maryland), western blots, immunoprecipitation assays, and immunohistochemistry.
  • amino acid sequence variants of the antibodies and/or the binding polypeptides provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody and/or binding polypeptide.
  • Amino acid sequence variants of an antibody and/or binding polypeptides may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody and/or binding polypeptide, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues within, the amino acid sequences of the antibody and/or binding polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., target-binding (GDF11, GDF8, ActRIIA, and/or ActRIIB binding).
  • target-binding GDF11, GDF8, ActRIIA, and/or ActRIIB binding
  • Alterations may be made in HVRs, for example, to improve antibody affinity.
  • Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury (2008) Methods Mol. Biol. 207:179-196 (2008 )), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity.
  • affinity maturation by constructing and reselecting from secondary libraries has been described in the art [see, e.g., Hoogenboom et al., in Methods in Molecular Biology 178:1-37, O'Brien et al., ed., Human Press, Totowa, N.J., (2001 )].
  • diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis).
  • a secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity.
  • HVR-directed approaches in which several HVR residues (e.g., 4-6 residues at a time) are randomized.
  • HVR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling.
  • CDR-H3 and CDR-L3 in particular are often targeted.
  • substitutions, insertions, or deletions may occur within one or more HVRs so long as such alterations do not substantially reduce the ability of the antibody to bind to the antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • Such alterations may be outside of HVR "hotspots" or SDRs.
  • each HVR either is unaltered, or contains no more than one, two, or three amino acid substitutions.
  • a useful method for identification of residues or regions of the antibody and/or the binding polypeptide that may be targeted for mutagenesis is called “alanine scanning mutagenesis", as described by Cunningham and Wells (1989) Science, 244:1081-1085 .
  • a residue or group of target residues e.g., charged residues such as arg, asp, his, lys, and glu
  • a neutral or negatively charged amino acid e.g., alanine or polyalanine
  • a crystal structure of an antigen-antibody complex can be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.
  • Amino-acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues.
  • terminal insertions include an antibody with an N-terminal methionyl residue.
  • Other insertional variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.
  • an antibody and/or binding polypeptide provided herein may be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody and/or binding polypeptide include but are not limited to water-soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody and/or binding polypeptide may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody and/or binding polypeptide to be improved, whether the antibody derivative and/or binding polypeptide derivative will be used in a therapy under defined conditions.
  • any of the ActRII antagonist antibodies disclosed herein e.g., an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIB antibody
  • an anti-activin A antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an anti-BMP7 antibody, an anti-ActRIIA antibody, and/or or an anti-ActRIIB antibody can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent
  • an ActRII antagonist antibody disclosed herein e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDF11 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody
  • an ActRII antagonist antibody disclosed herein can be used in combination with i) one or more additional ActRII antagonist antibodies disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF traps disclosed herein; iv) one or more small-molecule ActRII antagonist disclosed herein (e.g., a small molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP
  • the present disclosure relates to a small molecule, or combination of small molecules, that antagonizes ActRII activity (e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling).
  • ActRII activity e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling.
  • the disclosure provides methods of using a small-molecule antagonist or combination of small antagonists of ActRII, alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive therapies [ e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF), transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea, etc .], to, e.g., increase red blood cell levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, treat sickle-cell disease in a subject in need thereof, treat or prevent one or more complication of sickle-cell disease (e.g., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-oc
  • a preferred ActRII antagonist of the present disclosure is a small-molecule antagonist, or combination of small-molecule antagonists, that direct or indirect inhibits at least GDF11 activity.
  • a small-molecule antagonist, or combination of small-molecule antagonists may further inhibit, either directly or indirectly, GDF8.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure does not substantially inhibit activin A activity.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure that inhibits, either directly or indirectly, GDF11 and/or GDF8 activity further inhibits, either directly or indirectly, activity of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure is an indirect inhibitor of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, Nodal, ActRIIA, and ActRIIB.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure may inhibit the expression (e.g., transcription, translation, cellular secretion, or combinations thereof) of at least GDF11.
  • such a small-molecule antagonist, or combination of small-molecule antagonists may further inhibit expression of GDF8.
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure does not substantially inhibit the expression of activin A.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure that inhibits expression of GDF11 and/or GDF8 may further inhibit the expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure is direct inhibitor of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • a preferred small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure directly binds to and inhibits at least GDF11 activity (e.g. inhibits the ability GDF11 to bind to an ActRIIA and/or ActRIIB receptor; inhibits GDF11-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure may further bind to and inhibit GDF8 activity (e.g. inhibits the ability of GDF8 to bind to an ActRIIA and/or ActRIIB receptor; inhibits GDF8-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • GDF8 activity e.g. inhibits the ability of GDF8 to bind to an ActRIIA and/or ActRIIB receptor; inhibits GDF8-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling.
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure does not substantially bind to or inhibit activin A activity (e.g.
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure that binds to and inhibits the activity of GDF11 and/or GDF8 further binds to and inhibits the activity of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure directly binds to and inhibits at least GDF8 activity (e.g. inhibits the ability GDF8 to bind to an ActRIIA and/or ActRIIB receptor; inhibits GDF8-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure may further bind to and inhibit GDF11 activity (e.g.
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure does not substantially bind to or inhibit activin A activity (e.g. the ability of activin A to bind to an ActRIIA and/or ActRIIB receptor; activin A-mediated activation of the ActRIIA and/or ActRIIB signaling transduction, SMAD 2/3 signaling).
  • a small-molecule antagonist, or combinations of small-molecule antagonists, of the disclosure that binds to and inhibits the activity of GDF8 and/or GDF11 further binds to and inhibits the activity of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure directly binds to and inhibits at least ActRIIA activity (e.g. ActRII ligand-mediated activation of ActRIIA signaling transduction, such as SMAD 2/3 signaling).
  • a preferred small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure binds to an ActRIIA receptor and inhibits at least GDF11 from binding to and/or activating the ActRIIA receptor.
  • such a small-molecule antagonist, or combination of small-molecule antagonists may further inhibit GDF8 from binding to and/or activating the ActRIIA receptor.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure does not substantially inhibit activin A from binding to and/or activating an ActRIIA receptor.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure that inhibits GDF11 and/or GDF8 from binding to and/or activating the ActRIIA receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal from binding to/and or activating the ActRIIA receptor.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the present disclosure directly binds to and inhibits at least ActRIIB activity (e.g. ActRII ligand-mediated activation of ActRIIB signaling transduction, such as SMAD 2/3 signaling).
  • a preferred small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure binds to an ActRIIB receptor and inhibits at least GDF11 from binding to and/or activating the ActRIIB receptor.
  • such a small-molecule antagonist, or combination of small-molecule antagonists may further inhibit GDF8 from binding to and/or activating the ActRIIB receptor.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure does not substantially inhibit activin A from binding to and/or activating an ActRIIB receptor.
  • a small-molecule antagonist, or combination of small-molecule antagonists, of the disclosure that inhibits GDF11 and/or GDF8 from binding to and/or activating the ActRIIB receptor further inhibits one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, and Nodal from binding to/and or activating the ActRIIB receptor.
  • Binding organic small molecule antagonists of the present disclosure may be identified and chemically synthesized using known methodology (see, e.g., PCT Publication Nos. WO 00/00823 and WO 00/39585 ).
  • small-molecule antagonists of the disclosure are usually less than about 2000 daltons in size, alternatively less than about 1500, 750, 500, 250 or 200 daltons in size, wherein such organic small molecules that are capable of binding, preferably specifically, to a polypeptide as described herein ( e.g., GDF11, GDF8, ActRIIA, and ActRIIB).
  • Such small-molecule antagonists may be identified without undue experimentation using well-known techniques.
  • Binding organic small molecules of the present disclosure may be, for example, aldehydes, ketones, oximes, hydrazones, semicarbazones, carbazides, primary amines, secondary amines, tertiary amines, N-substituted hydrazines, hydrazides, alcohols, ethers, thiols, thioethers, disulfides, carboxylic acids, esters, amides, ureas, carbamates, carbonates, ketals, thioketals, acetals, thioacetals, aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates, aromatic compounds, heterocyclic compounds, anilines, alkenes, alkynes, diols, amino alcohols, oxazolidines, oxazolines, thiazolidines, thiazolines, enamines, sulfonamide
  • any of the small-molecule ActRII antagonists disclosed herein e.g., a small-molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB
  • a small-molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • a small-molecule ActRII antagonist disclosed herein e.g., a small-molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB
  • a small-molecule ActRII antagonist disclosed herein can be used in combination with i) one or more additional small molecule ActRII antagonists disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF traps disclosed herein;
  • one or more ActRII antagonist antibodies disclosed herein e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti
  • the present disclosure relates to a polynucleotide, or combination of polynucleotides, that antagonizes ActRII activity (e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling).
  • ActRII activity e.g., inhibition of ActRIIA and/or ActRIIB signaling transduction, such as SMAD 2/3 and/or SMAD 1/5/8 signaling.
  • the disclosure provides methods of using a polynucleotide ActRII antagonist or combination of polynucleotide ActRII antagonists, alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive therapies [ e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF), transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea, etc .], to, e.g., increase red blood cell levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, treat sickle-cell disease in a subject in need thereof, treat or prevent one or more complication of sickle-cell disease (e.g., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis
  • a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonists, of the present disclosure can be used to inhibit the activity and/or expression on one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB.
  • a polynucleotide ActRII antagonist, or combination of polynucleotide ActRII antagonists, of the disclosure is a GDF-ActRII antagonist.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, secretion, or combinations thereof) of at least GDF11.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists may further inhibit the activity and/or expression of GDF8.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of GDF11 and/or GDF8 may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, secretion, or combinations thereof) of at least GDF8.
  • such polynucleotide antagonist, or combination of polynucleotide antagonists may further inhibit the activity and/or expression of GDF11.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of GDF8 and/or GDF11 may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, secretion, or combinations thereof) of at least ActRIIA.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of ActRIIA may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, and/or ActRIIB.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure inhibits the activity and/or expression (e.g., transcription, translation, secretion, or combinations thereof) of at least ActRIIB.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure does not substantially inhibit the activity and/or expression of activin A.
  • a polynucleotide antagonist, or combination of polynucleotide antagonists, of the disclosure that inhibits the activity and/or expression of ActRIIB may further inhibit the activity and or expression of one or more of activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, and/or ActRIIA.
  • the polynucleotide antagonists of the present disclosure may be an antisense nucleic acid, an RNAi molecule [ e.g., small interfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)], an aptamer and/or a ribozyme.
  • RNAi molecule e.g., small interfering RNA (siRNA), small-hairpin RNA (shRNA), microRNA (miRNA)
  • siRNA small interfering RNA
  • shRNA small-hairpin RNA
  • miRNA microRNA
  • the nucleic acid and amino acid sequences of human GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB are known in the art and thus polynucleotide antagonists for use in accordance with methods of the present disclosure may be routinely made by the skilled artisan based on the knowledge in the
  • antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation.
  • Antisense techniques are discussed, for example, in Okano (1991) J. Neurochem. 56:560 ; Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988 ).
  • Triple helix formation is discussed in, for instance, Cooney et al. (1988) Science 241:456 ; and Dervan et al., (1991)Science 251:1300 .
  • the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
  • the antisense nucleic acids comprise a single-stranded RNA or DNA sequence that is complementary to at least a portion of an RNA transcript of a gene disclosed herein (e.g., GDF11, GDF8, activin A, activin B, activin C, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB).
  • GDF11, GDF8 activin A, activin B, activin C, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • absolute complementarity although preferred, is not required.
  • a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids of a gene disclosed herein (e.g., GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB), a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
  • the larger the hybridizing nucleic acid the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Polynucleotides that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
  • sequences complementary to the 3'-untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well [see, e.g., Wagner, R., (1994) Nature 372:333-335 ].
  • oligonucleotides complementary to either the 5'- or 3'-untranslated, noncoding regions of a gene of the disclosure e.g., GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB
  • Polynucleotides complementary to the 5'-untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense polynucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the methods of the present disclosure.
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.
  • the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.
  • the antisense nucleic acid of the present disclosure e.g., a GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, or ActRIIB antisense nucleic acid
  • a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of a gene of the disclosure.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the desired antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding desired genes of the instant disclosure, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive.
  • promoters include, but are not limited to, the SV40 early promoter region [see, e.g., Benoist and Chambon (1981) Nature 29:304-310 ], the promoter contained in the 3' long terminal repeat of Rous sarcoma virus [see, e.g., Yamamoto et al.
  • the polynucleotide antagonists are interfering RNA or RNAi molecules that target the expression of one or more of: GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB.
  • RNAi refers to the expression of an RNA which interferes with the expression of the targeted mRNA. Specifically, RNAi silences a targeted gene via interacting with the specific mRNA through a siRNA (small interfering RNA). The ds RNA complex is then targeted for degradation by the cell.
  • siRNA molecule is a double-stranded RNA duplex of 10 to 50 nucleotides in length, which interferes with the expression of a target gene which is sufficiently complementary (e.g. at least 80% identity to the gene).
  • the siRNA molecule comprises a nucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99, or 100% identical to the nucleotide sequence of the target gene.
  • RNAi molecules include short-hairpin RNA (shRNA); also short-interfering hairpin and microRNA (miRNA).
  • shRNA short-hairpin RNA
  • miRNA microRNA
  • the shRNA molecule contains sense and antisense sequences from a target gene connected by a loop. The shRNA is transported from the nucleus into the cytoplasm, and it is degraded along with the mRNA.
  • Pol III or U6 promoters can be used to express RNAs for RNAi.
  • Paddison et al. [Genes & Dev. (2002) 16:948-958, 2002 ] have used small RNA molecules folded into hairpins as a means to effect RNAi. Accordingly, such short hairpin RNA (shRNA) molecules are also advantageously used in the methods described herein.
  • stem lengths can range anywhere from about 25 to about 30 nt, and loop size can range between 4 to about 25 nt without affecting silencing activity. While not wishing to be bound by any particular theory, it is believed that these shRNAs resemble the double-stranded RNA (dsRNA) products of the DICER RNase and, in any event, have the same capacity for inhibiting expression of a specific gene.
  • the shRNA can be expressed from a lentiviral vector.
  • An miRNA is a single-stranded RNA of about 10 to 70 nucleotides in length that are initially transcribed as pre-miRNA characterized by a "stem-loop" structure and which are subsequently processed into mature miRNA after further processing through the RISC.
  • RNAi molecules that mediate RNAi, including without limitation siRNA
  • chemical synthesis Hohjoh, FEBS Lett 521:195-199, 2002
  • hydrolysis of dsRNA Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002
  • T7 RNA polymerase Donzeet et al., Nucleic Acids Res 30:e46, 2002 ; Yu et al., Proc Natl Acad Sci USA 99:6047-6052, 2002
  • hydrolysis of double-stranded RNA using a nuclease such as E. coli RNase III ( Yang et al., Proc Natl Acad Sci USA 99:9942-9947, 2002 ).
  • the disclosure provides polynucleotide antagonists including but not limited to, a decoy DNA, a double-stranded DNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA, a double-stranded RNA, a molecule capable of generating RNA interference, or combinations thereof.
  • a decoy DNA including but not limited to, a decoy DNA, a double-stranded DNA, a single-stranded DNA, a complexed DNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, a viral RNA, a double-stranded RNA, a molecule capable of generating RNA interference, or combinations thereof.
  • the polynucleotide antagonists of the disclosure are aptamers.
  • Aptamers are nucleic acid molecules, including double-stranded DNA and single-stranded RNA molecules, which bind to and form tertiary structures that specifically bind to a target molecule, such as a GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB polypeptide.
  • a target molecule such as a GDF11, GDF8, activin A, activin B, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and ActRIIB polypeptide.
  • the generation and therapeutic use of aptamers are well established in the art. See, e.g., U.S. Pat. No. 5,475,096 . Additional information on aptamers can be found in U.S. Patent Application Publication No.
  • Nucleic acid aptamers are selected using methods known in the art, for example via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process.
  • SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules as described in, e.g., U.S. Pat. Nos. 5,475,096 , 5,580,737 , 5,567,588 , 5,707,796 , 5,763,177 , 6,011,577 , and 6,699,843 .
  • Another screening method to identify aptamers is described in U.S. Pat. No. 5,270,163 .
  • the SELEX process is based on the capacity of nucleic acids for forming a variety of two- and three-dimensional structures, as well as the chemical versatility available within the nucleotide monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric, including other nucleic acid molecules and polypeptides. Molecules of any size or composition can serve as targets.
  • the SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve desired binding affinity and selectivity.
  • the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding; partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; dissociating the nucleic acid-target complexes; amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand enriched mixture of nucleic acids.
  • the steps of binding, partitioning, dissociating and amplifying are repeated through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
  • binding molecules are separately administered to the animal [see, e.g., O'Connor (1991) J. Neurochem. 56:560 ], but such binding molecules can also be expressed in vivo from polynucleotides taken up by a host cell and expressed in vivo [see, e.g., Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988 )].
  • any of the polynucleotide ActRII antagonists disclosed herein e.g., a polynucleotide antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB
  • a polynucleotide antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB can be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • an polynucleotide ActRII antagonist disclosed herein e.g., a polynucleotide antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB, activin C, activin E, BMP6, BMP7, Nodal, ActRIIA, and/or ActRIIB
  • a polynucleotide ActRII antagonist disclosed herein can be used in combination with i) one or more additional polynucleotide ActRII antagonists disclosed herein, ii) one or more ActRII polypeptides disclosed herein ( e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF traps disclosed herein;
  • one or more ActRII antagonist antibodies disclosed herein e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-
  • an agent for use in accordance with the methods disclosed herein is a follistatin polypeptide, which may be used alone or in combination with one or more erythropoiesis stimulating agents (e.g., EPO) or other supportive therapies [e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF), transfusion of red blood cells or whole blood, iron chelation therapy, treatment with hydroxyurea, etc.], to, increase red blood cell levels in a subject in need thereof, treat or prevent an anemia in a subject in need thereof, and methods of treating a sickle-cell disease in a subject in need thereof, and/or methods of treating or preventing one or more complications of sickle-cell disease), and/or reduce blood transfusion burden.
  • erythropoiesis stimulating agents e.g., EPO
  • supportive therapies e.g., hematopoietic growth factors (e.g., G-CSF or GM-CSF), transfusion of red blood
  • follistatin polypeptide includes polypeptides comprising any naturally occurring polypeptide of follistatin as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity, and further includes any functional monomer or multimer of follistatin.
  • follistatin polypeptides of the disclosure bind to and/or inhibit activin activity, particularly activin A (e.g., activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3 signaling).
  • activin A e.g., activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3 signaling.
  • Variants of follistatin polypeptides that retain activin binding properties can be identified based on previous studies involving follistatin and activin interactions.
  • WO2008/030367 discloses specific follistatin domains ("FSDs”) that are shown to be important for activin binding.
  • FSDs follistatin domains
  • SEQ ID NOs: 18-20 the follistatin N-terminal domain
  • FSD2 SEQ ID NO: 20
  • FSD1 SEQ ID NO: 19
  • methods for making and testing libraries of polypeptides are described above in the context of ActRII polypeptides, and such methods also pertain to making and testing variants of follistatin.
  • Follistatin polypeptides include polypeptides derived from the sequence of any known follistatin having a sequence at least about 80% identical to the sequence of a follistatin polypeptide, and optionally at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity.
  • follistatin polypeptides include the mature follistatin polypeptide or shorter isoforms or other variants of the human follistatin precursor polypeptide (SEQ ID NO: 16) as described, for example, in WO2005/025601 .
  • the human follistatin precursor polypeptide isoform FST344 is as follows:
  • the signal peptide is underlined ; also underlined above are the last 27 residues which represent the C-terminal extension distinguishing this follistatin isoform from the shorter follistatin isoform FST317 shown below.
  • the human follistatin precursor polypeptide isoform FST317 is as follows:
  • the signal peptide is underlined .
  • FSND follistatin N-terminal domain
  • the FSD1 and FSD2 sequences are as follows:
  • an agent for use in accordance with the methods disclosed herein is a follistatin-like related gene (FLRG), also known as follistatin-related protein 3 (FSTL3).
  • FLRG polypeptide includes polypeptides comprising any naturally occurring polypeptide of FLRG as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a useful activity.
  • FLRG polypeptides of the disclosure bind to and/or inhibit activin activity, particularly activin A (e.g., activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3 signaling).
  • activin A e.g., activin-mediated activation of ActRIIA and/or ActRIIB SMAD 2/3 signaling.
  • Variants of FLRG polypeptides that retain activin binding properties can be identified using routine methods to assay FLRG and activin interactions (see, e.g., US 6,537,966 ).
  • methods for making and testing libraries of polypeptides are described above in the context of ActRII polypeptides and such methods also pertain to making and testing variants of FLRG.
  • FLRG polypeptides include polypeptides derived from the sequence of any known FLRG having a sequence at least about 80% identical to the sequence of an FLRG polypeptide, and optionally at least 85%, 90%, 95%, 97%, 99% or greater identity.
  • the human FLRG precursor (follistatin-related protein 3 precursor) polypeptide is as follows:
  • the signal peptide is underlined .
  • follistatin polypeptides and FLRG polypeptides include fusion proteins having at least a portion of the follistatin polypeptide or FLRG polypeptide and one or more fusion domains, such as, for example, domains that facilitate isolation, detection, stabilization or multimerization of the polypeptide. Suitable fusion domains are discussed in detail above with reference to the ActRII polypeptides.
  • an antagonist agent of the disclosure is a fusion protein comprising an activin-binding portion of a follistatin polypeptide fused to an Fc domain.
  • an antagonist agent of the disclosure is a fusion protein comprising an activin binding portion of an FLRG polypeptide fused to an Fc domain.
  • follistatin polypeptides disclosed herein may be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • a follistatin polypeptide disclosed herein can be used in combination with i) one or more additional follistatin polypeptides disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF traps disclosed herein; iv) one or more ActRII antagonist antibodies disclosed herein (e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody); v) one or more small molecule ActRII antagonists disclosed herein (e.g., a small molecule antagonist of one or more of GDF11, GDF8, activin A, activin B
  • any of the FLRG polypeptides disclosed herein may be combined with one or more additional ActRII antagonist agents of the disclosure to achieve the desired effect (e.g., increase red blood cell levels and/or hemoglobin in a subject in need thereof, treat or prevent an anemia, treat sickle-cell disease, treat or prevent one or more complications of sickle-cell disease).
  • a FLRG polypeptide disclosed herein can be used in combination with i) one or more additional FLRG polypeptides disclosed herein, ii) one or more ActRII polypeptides disclosed herein (e.g., ActRIIA and/or ActRIIB polypeptides), iii) one or more GDF traps disclosed herein; iv) one or more ActRII antagonist antibodies disclosed herein (e.g., an anti-GDFl 1 antibody, an anti-activin B antibody, an anti-activin C antibody, an anti-activin E antibody, an anti-GDFl 1 antibody, an anti-GDF8 antibody, an anti-BMP6 antibody, an-anti-BMP7 antibody, an anti-ActRIIA antibody, or an anti-ActRIIB antibody); v) one or more small molecule ActRII antagonists disclosed herein (e.g., a small molecule antagonist of one or more of GDF11, GDF8, activin A, activin B, activin AB,
  • the present disclosure relates to the use of the subject ActRII polypeptides (e.g., ActRIIA and ActRIIB polypeptides) and GDF trap polypeptides to identify compounds (agents) which are agonist or antagonists of ActRIIB polypeptides.
  • Compounds identified through this screening can be tested to assess their ability to modulate red blood cell, hemoglobin, and/or reticulocyte levels in vivo or in vitro. These compounds can be tested, for example, in animal models.
  • ActRII signaling e.g., ActRIIA and/or ActRIIB SMAD 2/3 and/or SMAD 1/5/8 signaling.
  • high-throughput screening of compounds can be carried out to identify agents that perturb ActRII-mediated effects on a selected cell line.
  • the assay is carried out to screen and identify compounds that specifically inhibit or reduce binding of an ActRII polypeptide or GDF trap polypeptide to its binding partner, such as an ActRII ligand (e.g., activin A, activin B, activin AB, activin C, Nodal, GDF8, GDF11 or BMP7).
  • an ActRII ligand e.g., activin A, activin B, activin AB, activin C, Nodal, GDF8, GDF11 or BMP7.
  • the assay can be used to identify compounds that enhance binding of an ActRII polypeptide or GDF trap polypeptide to its binding partner such as an ActRII ligand.
  • the compounds can be identified by their ability to interact with an ActRII polypeptide or GDF trap polypeptide.
  • test compounds (agents) of the invention may be created by any combinatorial chemical method.
  • the subject compounds may be naturally occurring biomolecules synthesized in vivo or in vitro.
  • Compounds (agents) to be tested for their ability to act as modulators of tissue growth can be produced, for example, by bacteria, yeast, plants or other organisms ( e.g., natural products), produced chemically (e.g., small molecules, including peptidomimetics), or produced recombinantly.
  • Test compounds contemplated by the present invention include non-peptidyl organic molecules, peptides, polypeptides, peptidomimetics, sugars, hormones, and nucleic acid molecules.
  • the test agent is a small organic molecule having a molecular weight of less than about 2,000 Daltons.
  • test compounds of the disclosure can be provided as single, discrete entities, or provided in libraries of greater complexity, such as made by combinatorial chemistry.
  • libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds.
  • Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps.
  • the compounds may be optionally derivatized with other compounds and have derivatizing groups that facilitate isolation of the compounds.
  • Non-limiting examples of derivatizing groups include biotin, fluorescein, digoxygenin, green fluorescent protein, isotopes, polyhistidine, magnetic beads, glutathione S-transferase (GST), photoactivatible crosslinkers or any combinations thereof.
  • the effects of cellular toxicity or bioavailability of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect of the drug on the molecular target as may be manifest in an alteration of binding affinity between an ActRII polypeptide or a GDF trap polypeptide and its binding partner (e.g., an ActRII ligand).
  • the compound of interest is contacted with an isolated and purified ActRIIB polypeptide which is ordinarily capable of binding to an ActRIIB ligand, as appropriate for the intention of the assay.
  • an isolated and purified ActRIIB polypeptide which is ordinarily capable of binding to an ActRIIB ligand, as appropriate for the intention of the assay.
  • an ActRIIB ligand e.g., GDF11
  • Detection and quantification of ActRIIB/ActRIIB ligand complexes provides a means for determining the compound's efficacy at inhibiting (or potentiating) complex formation between the ActRIIB polypeptide and its binding protein.
  • the efficacy of the compound can be assessed by generating dose-response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • isolated and purified ActRIIB ligand is added to a composition containing the ActRIIB polypeptide, and the formation of ActRIIB/ActRIIB ligand complex is quantitated in the absence of the test compound.
  • the order in which the reactants may be admixed can be varied, and can be admixed simultaneously.
  • cellular extracts and lysates may be used to render a suitable cell-free assay system.
  • Complex formation between an ActRII polypeptide or GDF trap polypeptide and its binding protein may be detected by a variety of techniques. For instance, modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled (e.g., 32 P, 35 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzymatically labeled ActRII polypeptide or GDF trap polypeptide and/or its binding protein, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled (e.g., 32 P, 35 S, 14 C or 3 H), fluorescently labeled (e.g., FITC), or enzymatically labeled ActRII polypeptide or GDF trap polypeptide and/or its binding protein, by immunoassay, or by chromatographic detection.
  • the present disclosure contemplates the use of fluorescence polarization assays and fluorescence resonance energy transfer (FRET) assays in measuring, either directly or indirectly, the degree of interaction between an ActRII polypeptide of GDF trap polypeptide and its binding protein.
  • FRET fluorescence resonance energy transfer
  • other modes of detection such as those based on optical waveguides (see, e.g., PCT Publication WO 96/26432 and U.S. Pat. No. 5,677,196 ), surface plasmon resonance (SPR), surface charge sensors, and surface force sensors, are compatible with many embodiments of the disclosure.
  • an interaction trap assay also known as the "two-hybrid assay," for identifying agents that disrupt or potentiate interaction between an ActRII polypeptide or GDF trap polypeptide and its binding partner. See, e.g., U.S. Pat. No. 5,283,317 ; Zervos et al. (1993) Cell 72:223-232 ; Madura et al. (1993) J Biol Chem 268:12046-12054 ; Bartel et al. (1993) Biotechniques 14:920-924 ; and Iwabuchi et al. (1993) Oncogene 8:1693-1696 ).
  • the present disclosure contemplates the use of reverse two-hybrid systems to identify compounds (e.g., small molecules or peptides) that dissociate interactions between an ActRII polypeptide or GDF trap and its binding protein [see, e.g., Vidal and Legrain, (1999) Nucleic Acids Res 27:919-29 ; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81 ; and U.S. Pat. Nos. 5,525,490 ; 5,955,280 ; and 5,965,368 ].
  • compounds e.g., small molecules or peptides
  • the subject compounds are identified by their ability to interact with an ActRII polypeptide or GDF trap polypeptide.
  • the interaction between the compound and the ActRII polypeptide or GDF trap polypeptide may be covalent or noncovalent.
  • such interaction can be identified at the protein level using in vitro biochemical methods, including photo-crosslinking, radiolabeled ligand binding, and affinity chromatography [see, e.g., Jakoby WB et al. (1974) Methods in Enzymology 46:1 ].
  • the compounds may be screened in a mechanism-based assay, such as an assay to detect compounds which bind to an ActRII polypeptide of GDF trap polypeptide.
  • This may include a solid-phase or fluid-phase binding event.
  • the gene encoding an ActRII polypeptide or GDF trap polypeptide can be transfected with a reporter system (e.g., ⁇ -galactosidase, luciferase, or green fluorescent protein) into a cell and screened against the library preferably by high-throughput screening or with individual members of the library.
  • a reporter system e.g., ⁇ -galactosidase, luciferase, or green fluorescent protein
  • Other mechanism-based binding assays may be used; for example, binding assays which detect changes in free energy. Binding assays can be performed with the target fixed to a well, bead or chip or captured by an immobilized antibody or resolved by capillary electrophoresis. The bound compounds may be detected usually using colorimetric endpoints or fluorescence or surface plasmon resonance.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used to increase red blood cell levels in a subject in need thereof, particularly mammals such as rodents, primates, and humans.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used to treat or prevent an anemia in a subject in need thereof, particularly mammals such as rodents, primates, and humans.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used treat sickle-cell disease, particularly mammals such as rodents, primates, and humans.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used treat or prevent anemia in a sickle-cell disease subject in need thereof, particularly mammals such as rodents, primates, and humans.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used treat or prevent one or more complications of sickle-cell disease (e.g., anemia, anemia crisis, splenomegaly, pain crisis, chest syndrome, acute chest syndrome, blood transfusion requirement, organ damage, pain medicine (management) requirement, splenic sequestration crises, hyperhemolytic crisis, vaso-occlusion, vaso-occlusion crisis, acute myocardial infarction, sickle-cell chronic lung disease, thromboemboli, hepatic failure, hepatomegaly, hepatic sequestration, iron overload, splenic infarction, acute and/or chronic renal failure, pyelonephritis
  • sickle-cell disease
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used treat or prevent vaso-occlusion crisis in a sickle-cell disease subject in need thereof, particularly mammals such as rodents, primates, and humans.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used treat or prevent pain crisis in a sickle-cell disease subject in need thereof, particularly mammals such as rodents, primates, and humans.
  • an ActRII antagonist agent, or combination of ActRII antagonist agents, of the present disclosure can be used treat or prevent an anemia crisis in a sickle-cell disease subject in need thereof, particularly mammals such as rodents, primates, and humans.
  • a therapeutic that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • treating includes amelioration or elimination of the condition once it has been established. In either case, prevention or treatment may be discerned in the diagnosis provided by a physician or other health care provider and the intended result of administration of the therapeutic agent.
  • treatment or prevention of a disease or condition as described in the present disclosure is achieved by administering one or more of the ActRII antagonists (e.g., an ActRIIA and/or ActRIIB antagonist) of the present disclosure in an effective amount.
  • An effective amount of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a “therapeutically effective amount” of an agent of the present disclosure may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
  • sickle-cell disease is an inherited disorder caused by a mutation in the ⁇ -globin gene (a mutation of a glutamate to a valine at codon 6). See, e.g., Kassim et al. (2013) Annu Rev Med, 64: 451-466 .
  • Sickle-cell anemia refers to the most common form of sickle-cell disease, with a homozygous mutation in the ⁇ S allele (HbSS), affecting 60 to 70% of people with sickle-cell disease.
  • abnormal hemoglobin molecules are produced with a hydrophobic motif that is exposed when it is in a deoxygenated state [see, e.g., Eaton et al. (1990) Adv Protein Chem, 40: 63-279 ; Steinberg, MH (1999) N Engl J Med 340(13): 1021-1030 ; and Ballas et al. (1992) Blood, 79(8): 2154-63 ].
  • the chains of the separate hemoglobin molecules polymerize, which results in damage to the red blood cell membrane and cellular dehydration.
  • the membrane damage is manifested, in part, by a redistribution of membrane lipids leading to the expression of phosphatidylserine on the outer leaflet of the erythrocyte membrane [see, e.g., ( 2002) Blood 99(5): 1564-1571 ].
  • Externalized phosphatidylserine promotes adhesion to both macrophages and activated endothelial cells, which contributes to vascular (vaso) occlusion.
  • the red cell's hemoglobin precipitates into long crystals that cause it to elongate, morphologically switching into a "sickled" red blood cell.
  • Both genotype and the extent and degree of deoxygenation contribute to the severity of hemoglobin polymerization. It has been demonstrated that the presence of fetal hemoglobin proportionally reduces the amount of pathological hemoglobin polymers and is protective from vaso-occlusive crises.
  • a sickle-cell pain crisis refers to acute sickling-related pain that lasts for at least 1 hour ( e.g., at least 1, 2, 3, 4, 5, 6, or 10 hours) and optionally requires pain management therapy such as, e.g., administration of one or more narcotic and/or non-steroid anti-inflammatory agent.
  • a pain crisis typically results in patient admission to a medical facility for pain management therapy.
  • Acute pain in patients with sickle-cell disease is generally ischemic in nature and can result from the occlusion of microvascular beds.
  • Clinical data indicate that some patients with sickle-cell disease have from three to ten episodes of pain crisis per year. In many patients a pain-crisis episode will typically be resolved in about a week.
  • one or more opioid analgesics e.g. hydromorphone, meperidine, etc .
  • non-steroidal anti-inflammatory drugs e.g., ketorolac tromethamine
  • corticosteroids e.g., corticosteroids
  • one or more ActRII antagonist agents of the disclosure optionally combined with an EPO receptor activator and/or one or more additional therapies (e.g., treatment with hydroxyurea), may be used to treat or prevent pain crisis in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure may be used to reduce the frequency of pain management (e.g., treatment with one or more narcotics, non-steroid anti-inflammatory drugs, and/or corticosteroids) in a patient with sickle-cell disease.
  • pain management e.g., treatment with one or more narcotics, non-steroid anti-inflammatory drugs, and/or corticosteroids
  • one or more ActRII antagonist agents of the disclosure may be used to reduce the dosage amount of one or more pain management agents (e.g., narcotics, non-steroid anti-inflammatory drugs, and/or corticosteroids) in a patient with sickle-cell disease.
  • pain management agents e.g., narcotics, non-steroid anti-inflammatory drugs, and/or corticosteroids
  • one or more ActRII antagonist agents of the disclosure may be used to reduce the frequency of blood transfusions or reduce the dosage of chelation therapy in SCD patients.
  • one or more ActRII antagonist agents of the disclosure may be used to reduce liver iron content and/or prevent or reverse a hepatic complication of iron overload including, e.g., liver enlargement (hepatomegaly), liver fibrosis (increase in scar tissue), and cirrhosis (extensive scarring).
  • one or more ActRII antagonist agents of the disclosure may be used to prevent or reverse an endocrine complication of iron overload including, e.g., diabetes mellitus.
  • Vaso-occlusive crises are one of the clinical hallmarks of sickle-cell disease. See, e.g., Rees et al. (2010) Lancet, 376: 2018-2031 .
  • Hypoxia, acidosis, inflammatory stress, and endothelial cell activation promote the entrapment of rigid, polymerized sickled erythrocytes and leukocytes within small vessels.
  • Sickled red blood cells obstruct capillaries and restrict blood flow to the organ, leading to ischemia, pain, tissue necrosis, and damage to various organs. This can cause vascular obstruction, leading to tissue ischemia.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent vaso-occlusive crisis in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent vaso-occlusion in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent a complication of vaso-occlusion in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • an EPO receptor activator and/or one or more additional therapies e.g., treatment with hydroxyurea
  • additional therapies e.g., treatment with hydroxyurea
  • hemolytic anemia leads to significant morbidity in patients with sickle-cell disease. See, e.g., Pakbaz et al. (2014) Hematol Oncol Clin N Am 28: 355-374 ; Kassim et al. (2013) Annu Rev Med 64: 451-466 .
  • Multiple factors contribute to chronic anemia in sickle-cell disease. As erythrocytes become deformed, antibodies are created to exposed antigens, which leads to increased destruction of erythrocytes, with an average lifespan of 17 days instead of 110 to 120 days. The release of hemoglobin during hemolysis inhibits nitric oxide signaling, leading to endothelial cell dysfunction and contributing to a hypercoagulable state.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent anemia in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent a complication of anemia in a sickle-cell disease patient.
  • Acute anemia which can be severe and potentially fatal, is associated with a 10% to 15% mortality rate in patients with sickle-cell disease.
  • severe episodes are precipitated by three main causes: splenic sequestration crises, aplastic crises, or hyperhemolytic crises [see, e.g., Ballas et al. (2010) Am J Hematol, 85: 6-13 ].
  • Splenic sequestration crises occur as a result of erythrocyte vaso-occlusion within the spleen, where a pooling of erythrocytes causes its rapid enlargement. As such, there is a decrease in circulating hemoglobin ( e.g., decreasing by 2 g/dL) and effective circulating volume, which may lead to hypovolemic shock.
  • one or more ActRII antagonist agents of the disclosure optionally in combination with one or more agents and/or supportive therapies for treating sickle-cell disease, may be used to treat or prevent splenic sequestration crises in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent splenic sequestration of red blood cells in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • an EPO receptor activator and/or one or more additional therapies e.g., treatment with hydroxyurea
  • additional therapies e.g., treatment with hydroxyurea
  • Aplastic crises arise when erythropoiesis is impaired. Because of the constant overproduction of erythrocytes, an aplastic crisis can rapidly result in severe anemia. Infections, such as parvovirus B19, streptococci, salmonella, and Epstein-Barr virus, are common causes for the transient arrest of erythropoiesis. Circulating erythrocytes and reticulocytes are both decreased during aplastic crises.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent aplastic crises in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • an EPO receptor activator and/or one or more additional therapies e.g., treatment with hydroxyurea
  • Hyperhemolysis occurs when there is a sudden exacerbation of anemia with reticulocytosis, without evidence of splenic sequestration. Hyperhemolytic crises have been documented in patients with multiple transfusions or in patients receiving intravenous immunoglobulin therapy.
  • one or more ActRII antagonist agents of the disclosure optionally in combination with one or more agents and/or supportive therapies for treating sickle-cell disease, may be used to treat or prevent hyperhemolytic crises in a sickle-cell disease patient.
  • one or more ActRII antagonist agents of the disclosure optionally in combination with one or more agents and/or supportive therapies for treating sickle-cell disease, may be used to treat or prevent hyperhemolytic anemia in a sickle-cell disease patient.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a cardiac complication of sickle-cell disease.
  • chronic anemia in sickle-cell disease causes a compensatory increased cardiac output. This, in turn, leads to cardiomegaly and left ventricular hypertrophy with left ventricular dysfunction. See, e.g., Adebayo et al. (2002) Niger J Med, 11: 145-152 ; Sachdev et al. (2007) J Am Coll Cardiol, 49: 472-279 ; and Zilberman et al. (2007) Am J Hematol 82: 433-438 .
  • Acute myocardial infarction can occur, even without coronary artery disease, and is thus underdiagnosed in sickle-cell disease [see, e.g., Pannu et al. (2008) Crit Pathw Cardio, 7: 133-138 ].
  • Cardiac arrhythmias and congestive heart failure have also been linked to premature death in sickle-cell disease patients [see, e.g., Fitzhugh et al. (2010) Am J Hematol 85: 36-40 ].
  • ActRII antagonist agents of the disclosure may be used to treat or prevent one or more cardiac complications of sickle-cell disease including, e.g., increased cardiac output, cardiomegaly, cardiomyopathy, left ventricular hypertrophy, acute myocardial infarction, arrhythmia, and congestive heart failure.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a pulmonary complication of sickle-cell disease.
  • Sickle-cell disease frequently results in both acute and chronic pulmonary complications [see, e.g., Rucknagel, DL (2001) Pediatr Pathol MOl Med, 20: 137-154 ; Haynes et al. (1986) Am J Med 80: 833-840 ].
  • Acute complications may include infection, pulmonary emboli from thrombi, bone marrow infarction, and fat emboli.
  • Pulmonary dysfunction may occur because of local pain from rib and sternal infarctions, leading to hypoventilation and atelectasis with hypoxemia.
  • Chronic complications include sickle-cell chronic lung disease and pulmonary hypertension.
  • Acute chest syndrome (ACS) is unique to people with sickle disease and is defined by a new pulmonary infiltrate involving at least one complete lung segment, chest pain, and temperature above 38.5°C along with tachypnea, wheeze, or cough [see, e.g., Vichinsky et al. (2000) N Engl J Med, 342: 1855-1865 ].
  • Development of pulmonary infarction, fat embolism, and infections may all contribute to ACS, and infection is a major cause of morbidity and mortality in patients with ACS.
  • Pulmonary hypertension is currently a major cause of morbidity and mortality in sickle-cell disease. See, e.g., De Castro et al. (2008) Am J Hematol, 83: 19-25 ; Gladwin et al. (2004) N Engl J Med 350: 886-895 . Pulmonary hypertension has been documented in 32% of adults with sickle-cell disease and is related to vaso-occlusive crises and hemolysis [see, e.g., Machado et al. (2010) Chest, 137(6 supple): 30S-38S ].
  • ActRII antagonist agents of the disclosure may be used to treat or prevent one or more pulmonary complications of sickle-cell disease including, e.g., fat or bone marrow emboli, pulmonary edema, sickle-cell lung disease, pulmonary hypertension, thromboemboli, and acute chest syndrome.
  • pulmonary complications of sickle-cell disease including, e.g., fat or bone marrow emboli, pulmonary edema, sickle-cell lung disease, pulmonary hypertension, thromboemboli, and acute chest syndrome.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a hepatic complication of sickle-cell disease.
  • Liver pathology is common in sickle-cell disease, with hepatomegaly being observed in ⁇ 90% of autopsy cases [see, e.g., Bauer et al. (1980) Am J med 69: 833-837 ; Mills et al. (1988) Arch Pathol Lab Med 112: 290-294 ].
  • the effects of sickle-cell anemia on the liver include intrasinusoidal sickling with proximal sinusoidal dilation, Kupffer cell hyperplasia with erythrophagocytosis, and hemosiderosis. Focal necrosis, regenerative nodules, and cirrhosis have also been described in postmortem examinations. Vaso-occlusion can lead to sinusoidal obstruction and ischemia, resulting in acute sickle hepatic crises. Similar to splenic sequestration, erythrocytes can be sequestered within the liver, leading to acute anemia [see, e.g., Lee et al. (1996) Postgrad Med J 72: 487-488 ].
  • Hepatic sequestration can also lead to intrahepatic cholestasis [see, e.g., Shao et al. (1995) Am J Gastroenterol 90: 2045-2050 ].
  • Ischemia within hepatocytes from sickling episodes also leads to ballooning of erythrocytes and intracanalicular cholestasis.
  • Some therapies used for treating sickle-cell disease also contribute to liver pathology. For example, frequent transfusions lead to increased iron deposition within Kupffer cells (which may lead to iron overload) and increase the risk of infection with blood-borne disease such as viral hepatitis.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent one or more hepatic complications of sickle-cell disease including, e.g., hepatic failure, hepatomegaly, hepatic sequestration, intrahepatic cholestasis, cholelithiasis, and iron overload.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a splenic complication of sickle-cell disease.
  • Splenic sequestration occurs as a result of vaso-occlusion of erythrocytes within the spleen. Acute exacerbations result in splenomegaly and occasionally splenic infarction. More commonly, subclinical splenic sequestration may lead to the gradual loss of splenic function, leading to functional hyposplenia and asplenia. This, in turn, can lead to an increased susceptibility to sepsis as a result of encapsulated bacteria.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent one or more splenic complications of sickle-cell disease including, e.g., acute or chronic splenic sequestration, splenomegaly, hyposplenia, asplenia, and splenic infarction.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a renal complication of sickle-cell disease.
  • Vaso-occlusion within the vasa recta capillaries leads to microthrombotic infarction and extravasation of erythrocytes into the renal medulla.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent one or more renal complications of sickle-cell disease including, e.g., acute and/or chronic renal failure, pyelonephritis, and renal medullary carcinoma.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a bone and/or joint complication of sickle-cell disease.
  • Bone and joint complications are a common complication in sickle-cell disease patients [see, e.g., Hernigou et al. (1991) J Bone Join Surg Am, 73: 81-92 ]. Pain from the small bones in the hands and feet, dactylitis, occurs frequently in infants with sickle-cell disease. Long-term consequences of vaso-occlusion within bone marrow include infarcts, necrosis, and ultimately degenerative changes.
  • Infarcted bone and bone marrow are common sites of infection, leading to osteomyelitis and septic arthritis. Osteonecrosis, or avascular necrosis, occurs after infarction with bone and bone marrow. Infarctions are most common within long bones such as the humerus, tibia, and femur. Chronic weight bearing causes stress on abnormal femoral heads and leads to progressive joint destruction and arthritis.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent one or more bone and/or joint complications of sickle-cell disease including, e.g., infarction, necrosis, osteomyelitis, septic arthritis, osteonecrosis, and osteopenia.
  • ActRII antagonist agents of the disclosure may be used to treat or prevent a neurological complication of sickle-cell disease.
  • Approximately 25 percent of individuals with sickle-cell disease are affected by neurological injury [see, e.g., Ohene-Frempong et al. (1998) Blood, 91: 288-294 ; Verduzco et al. (2009) Blood 114: 5117-5125 ].
  • the injuries may be acute or chronic. Cerebrovascular accidents are most common in adults, but depend on the genotype. A person with HbSS has the highest cerebrovascular risk, with a 24 percent likelihood of having a clinical stroke by the age of 45.
  • Ischemic strokes are more common in children under 9 years of age, whereas hemorrhagic strokes are more common in adults. Ischemic strokes occur because of the occlusion of large intracranial arteries, leading to ischemia.
  • the ischemia is secondary to occlusion of smaller vessels by rigid erythrocytes, exacerbated by chronic anemia, a hypercoagulable state, and flow-related hemodynamic injury to the arterial endothelium, further increasing the likelihood of erythrocyte adhesion.
  • hemorrhagic strokes may occur in intraventricular, intraparenchymal, and subarachnoid spaces [see, e.g., Anson, et al. (1991) J Neurosurg, 75: 552-558 ].
  • Intraventricular hemorrhage may be associated with rupture of anterior cerebral artery aneurysms or direct extension of intraparenchymal hemorrhage into the lateral or third ventricle.
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • additional therapies e.g., treatment with hydroxyurea
  • ActRII antagonist agents of the disclosure may be used to treat or prevent an ophthalmic complication of sickle-cell disease.
  • Eye complications in sickle-cell disease mainly affect the retina [see, e.g., Downes et al. (2005) Opthalmology, 112: 1869-1875 ; Fadugbagbe et al. (2010) Ann Trop Paediatr 30: 19-26 ].
  • New blood vessels (sea-fan formations) form mostly near arteriovenous crossings and are known as proliferative sickle retinopathy.
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • additional therapies e.g., treatment with hydroxyurea
  • ActRII antagonist agents of the disclosure may be administered to a subject in need thereof in combination with one or more additional agents (e.g., hydroxyurea, an EPO antagonist, EPO, an opioid analgesic, a non-steroidal anti-inflammatory drug, a corticosteroid, an iron-chelating agent) or supportive therapies (e.g., red blood cell transfusion) for treating sickle-cell disease or one or more complications of sickle-cell disease.
  • additional agents e.g., hydroxyurea, an EPO antagonist, EPO, an opioid analgesic, a non-steroidal anti-inflammatory drug, a corticosteroid, an iron-chelating agent
  • supportive therapies e.g., red blood cell transfusion
  • the mainstay of treatment for the majority of patients with sickle-cell disease is supportive.
  • Current treatment options for patients with sickle-cell disease include antibiotics, pain management, intravenous fluids, blood transfusion, surgery, and compounds such as hydroxyurea.
  • Hydroxyurea (e.g. Droxia®)is an approved drug for treating sickle-cell disease. Hydroxyurea is an S-phase cytotoxic drug and is used for long-term therapy. It is believed to increase the levels of hemoglobin F which prevents formation of S-polymers and red cell sickling. It is also believed to increase NO production.
  • a multi-center trial of hydroxyurea in adults with sickle-cell disease showed that hydroxyurea reduced the incidence of painful episodes by nearly half. However, presently hydroxyurea is used only in patients who suffer severe complications of sickle-cell disease and who are capable of following the daily dosage regimes. The general belief is that hydroxyurea therapy is effective only if given in a structured environment with a high potential for compliance.
  • the methods of the present disclosure relate to treating sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and hydroxyurea. In some embodiments, the methods of the present disclosure relate to treating or preventing one or more complications of sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and hydroxyurea.
  • one or more ActRII antagonist agents of the disclosure may be used in combination with transfusion of either red blood cells or whole blood to treat anemia in patients with sickle-cell disease or one or more complications of sickle-cell disease.
  • additional therapies e.g., treatment with hydroxyurea
  • the methods of the present disclosure relate to treating sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and one or more blood cell transfusions. In some embodiments, the methods of the present disclosure relate to treating or preventing one or more complications of sickle-cell disease in a subject in need thereof by administering a combination of an ActRII antagonist of the disclosure and one or more red blood cell transfusions.
  • treatment with one or more ActRII antagonists of the disclosure is effective at decreasing the transfusion requirement in a patient with sickle-cell disease, e.g., reduces the frequency and/or amount of blood transfusion required to effectively treat sickle-cell disease or one or more complications of sickle-cell disease.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • an EPO receptor activator and/or one or more additional therapies e.g., treatment with hydroxyurea
  • one or more iron-chelating molecules may be used in combination with one or more iron-chelating molecules to promote iron excretion in the urine and/or stool and thereby prevent or reverse tissue iron overload in SCD patients.
  • Effective iron-chelating agents should be able to selectively bind and neutralize ferric iron, the oxidized form of non-transferrin bound iron which likely accounts for most iron toxicity through catalytic production of hydroxyl radicals and oxidation products [see, e.g., Esposito et al. (2003) Blood 102:2670-2677 ]. These agents are structurally diverse, but all possess oxygen or nitrogen donor atoms able to form neutralizing octahedral coordination complexes with individual iron atoms in stoichiometries of 1:1 (hexadentate agents), 2:1 (tridentate), or 3:1 (bidentate) [ Kalinowski et al. (2005) Pharmacol Rev 57:547-583 ].
  • iron-chelating agents also are relatively low molecular weight (e.g., less than 700 daltons), with solubility in both water and lipids to enable access to affected tissues.
  • specific examples of iron-chelating molecules include deferoxamine (also known as desferrioxamine B, desferoxamine B, DFO-B, DFOA, DFB, or desferal), a hexadentate agent of bacterial origin requiring daily parenteral administration, and the orally active synthetic agents deferiprone (bidentate; also known as FerriproxTM) and deferasirox (tridentate; also known as bis-hydroxyphenyl-triazole, ICL670, or ExjadeTM).
  • Combination therapy consisting of same-day administration of two iron-chelating agents shows promise in patients unresponsive to chelation monotherapy and also in overcoming issues of poor patient compliance with dereroxamine alone [ Cao et al. (2011) Pediatr Rep 3(2):e17 ; and Galanello et al. (2010) Ann NY Acad Sci 1202:79-86 ].
  • one or more ActRII antagonist agents of the disclosure may be used to increase red blood cell, hemoglobin, or reticulocyte levels in healthy individuals and selected patient populations.
  • appropriate patient populations include those with undesirably low red blood cell or hemoglobin levels, such as patients with anemia or sickle-cell disease and those at risk for developing undesirably low levels of red blood cells or hemoglobin, such as patients about to undergo major surgery or other procedures that may result in substantial blood loss.
  • a patient with adequate red blood cell levels is treated with one or more ActRII antagonist agents to increase red blood cell levels, and then blood is withdrawn and stored for later use in transfusions.
  • One or more ActRII antagonist agents of the disclosure may be used to increase red blood cell levels, hemoglobin levels, and/or hematocrit levels in a patient having an anemia (e.g., a sickle-cell patient).
  • anemia e.g., a sickle-cell patient.
  • a level of less than normal for the appropriate age and gender category may be indicative of anemia, although individual variations are taken into account.
  • a hemoglobin level from 10-12.5 g/dl, and typically about 11.0 g/dl is considered to be within the normal range in healthy adults, although, in terms of therapy, a lower target level may cause fewer cardiovascular side effects. See, e.g., Jacobs et al. (2000) Nephrol Dial Transplant 15, 15-19 .
  • hematocrit levels (percentage of the volume of a blood sample occupied by the cells) can be used as a measure for anemia. Hematocrit levels for healthy individuals range from about 41-51% for adult males and from 35-45% for adult females.
  • a patient may be treated with a dosing regimen intended to restore the patient to a target level of red blood cells, hemoglobin, and/or hematocrit.
  • a dosing regimen intended to restore the patient to a target level of red blood cells, hemoglobin, and/or hematocrit.
  • hemoglobin and hematocrit levels vary from person to person, optimally, the target hemoglobin and/or hematocrit level can be individualized for each patient.
  • Anemia is frequently observed in patients having a tissue injury, an infection, and/or a chronic disease, particularly cancer. In some subjects, anemia is distinguished by low erythropoietin levels and/or an inadequate response to erythropoietin in the bone marrow. See, e.g., Adamson, 2008, Harrison's Principles of Internal Medicine, 17th ed.; McGraw Hill, New York, pp 628-634 .
  • Potential causes of anemia include, for example, blood-loss, nutritional deficits (e.g. reduced dietary intake of protein), medication reaction, various problems associated with the bone marrow, and many diseases.
  • anemia has been associated with a variety of disorders and conditions that include, for example, bone marrow transplantation; solid tumors (e.g., breast cancer, lung cancer, and colon cancer); tumors of the lymphatic system (e.g., chronic lymphocyte leukemia, non-Hodgkin's lymphoma, and Hodgkin's lymphoma); tumors of the hematopoietic system (e.g., leukemia, a myelodysplastic syndrome and multiple myeloma); radiation therapy; chemotherapy ( e.g., platinum containing regimens); inflammatory and autoimmune diseases, including, but not limited to, rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythematosis (SLE), acute or chronic skin diseases (e.g., psoriasis), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); acute or chronic renal disease or failure, including
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc.
  • an EPO receptor activator and/or one or more other additional therapies e.g., treatment with hydroxyurea
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent a cancer-related anemia.
  • hypoproliferative anemia can result from primary dysfunction or failure of the bone marrow.
  • Hypoproliferative anemias include: anemia of chronic disease, anemia of kidney disease, anemia associated with hypometabolic states, and anemia associated with cancer. In each of these types, endogenous erythropoietin levels are inappropriately low for the degree of anemia observed.
  • Other hypoproliferative anemias include: early-stage iron-deficient anemia, and anemia caused by damage to the bone marrow. In these types, endogenous erythropoietin levels are appropriately elevated for the degree of anemia observed. Prominent examples would be myelosuppression caused by cancer and/or chemotherapeutic drugs or cancer radiation therapy.
  • Myelosuppressive drugs include, for example: 1) alkylating agents such as nitrogen mustards (e.g., melphalan) and nitrosoureas ( e.g., streptozocin); 2) antimetabolites such as folic acid antagonists ( e.g., methotrexate), purine analogs ( e.g., thioguanine), and pyrimidine analogs ( e.g., gemcitabine); 3) cytotoxic antibiotics such as anthracyclines ( e.g., doxorubicin); 4) kinase inhibitors ( e.g., gefitinib); 5) mitotic inhibitors such as taxanes ( e.g., paclitaxel) and vinca alkaloids ( e.g., vinorelbine); 6) monoclonal antibodies ( e.g., rituximab); and 7) topoisomerase inhibitors (e.g., topotecan and
  • anemia can occur in Addison's disease, hypothyroidism, hyperparathyroidism, or males who are castrated or treated with estrogen.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • an EPO receptor activator may be used to treat or prevent a hyperproliferative anemia.
  • Chronic kidney disease is sometimes associated with hypoproliferative anemia, and the degree of the anemia varies in severity with the level of renal impairment. Such anemia is primarily due to inadequate production of erythropoietin and reduced survival of red blood cells.
  • Chronic kidney disease usually proceeds gradually over a period of years or decades to end-stage (Stage-5) disease, at which point dialysis or kidney transplantation is required for patient survival.
  • Anemia often develops early in this process and worsens as disease progresses.
  • the clinical consequences of anemia of kidney disease are well-documented and include development of left ventricular hypertrophy, impaired cognitive function, reduced quality of life, and altered immune function [see, e.g., Levin et al.
  • one or more ActRII antagonist agents of the disclosure may be used to treat or prevent anemia associated with acute or chronic renal disease or failure.
  • Anemia resulting from acute blood loss of sufficient volume is known as acute post-hemorrhagic anemia.
  • Acute blood loss initially causes hypovolemia without anemia since there is proportional depletion of RBCs along with other blood constituents.
  • hypovolemia will rapidly trigger physiologic mechanisms that shift fluid from the extravascular to the vascular compartment, which results in hemodilution and anemia. If chronic, blood loss gradually depletes body iron stores and eventually leads to iron deficiency.
  • one or more ActRII antagonist agents of the disclosure may be used to treat anemia resulting from acute blood loss.
  • a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc . may be used to treat anemia resulting from acute blood loss.
  • Iron-deficiency anemia is the final stage in a graded progression of increasing iron deficiency which includes negative iron balance and iron-deficient erythropoiesis as intermediate stages. Iron deficiency can result from increased iron demand, decreased iron intake, or increased iron loss, as exemplified in conditions such as pregnancy, inadequate diet, intestinal malabsorption, acute or chronic inflammation, and acute or chronic blood loss.
  • one or more ActRII antagonist agents of the disclosure may be used to treat a chronic iron-deficiency.
  • a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc . may be used to treat a chronic iron-deficiency.
  • MDS Myelodysplastic syndrome
  • MDS disorders include, for example, refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, refractory cytopenia with multilineage dysplasia, and myelodysplastic syndrome associated with an isolated 5q chromosome abnormality.
  • MDS patients are afflicted with chronic anemia. Therefore, MDS patients eventually require blood transfusions and/or treatment with growth factors (e.g., erythropoietin or G-CSF) to increase red blood cell levels.
  • growth factors e.g., erythropoietin or G-CSF
  • G-CSF erythropoietin
  • many MDS patients develop side-effect due to frequency of such therapies. For example, patients who receive frequent red blood cell transfusion can have tissue and organ damage from the buildup of extra iron.
  • one or more ActRII antagonist agents of the disclosure may be used to treat patients having MDS.
  • patients suffering from MDS may be treated using a one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .), optionally in combination with an EPO receptor activator.
  • patient suffering from MDS may be treated using a combination of one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc .) and one or more additional therapeutic agents for treating MDS including, for example, thalidomide, lenalidomide, azacitadine, decitabine, erythropoietins, deferoxamine, antithymocyte globulin, and filgrastrim (G-CSF).
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF Trap, etc .
  • additional therapeutic agents for treating MDS including, for example, thalidomide, lenalidomide, azacitadine, decitabine, erythropoietins, deferoxamine, anti
  • in combination with or “conjoint administration” refers to any form of administration such that additional therapies (e.g., second, third, fourth, etc .) are still effective in the body (e.g., multiple compounds are simultaneously effective in the patient, which may include synergistic effects of those compounds). Effectiveness may not correlate to measurable concentration of the agent in blood, serum, or plasma.
  • the different therapeutic compounds can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially, and on different schedules. Thus, an individual who receives such treatment can benefit from a combined effect of different therapies.
  • GDF11 and/or activin B antagonist agents can be administered concurrently with, prior to, or subsequent to, one or more other additional agents or supportive therapies.
  • each therapeutic agent will be administered at a dose and/or on a time schedule determined for that particular agent.
  • the particular combination to employ in a regimen will take into account compatibility of the antagonist of the present disclosure with the therapy and/or the desired therapeutic effect to be achieved.
  • ActRII antagonist agents of the disclosure may be used in combination with hepcidin or a hepcidin agonist for treating sickle-cell disease, particularly sickle-cell disease complications associated with iron overload.
  • hepcidin A circulating polypeptide produced mainly in the liver, hepcidin is considered a master regulator of iron metabolism by virtue of its ability to induce the degradation of ferroportin, an iron-export protein localized on absorptive enterocytes, hepatocytes, and macrophages.
  • hepcidin reduces availability of extracellular iron, so hepcidin agonists may be beneficial in the treatment of sickle-cell disease, particularly sickle-cell disease complications associated with iron overload.
  • One or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • an EPO receptor activator would also be appropriate for treating anemias of disordered RBC maturation, which are characterized in part by undersized (microcytic), oversized (macrocytic), misshapen, or abnormally colored (hypochromic) RBCs.
  • the present disclosure provides methods of treating or preventing anemia in an individual in need thereof by administering to the individual a therapeutically effective amount of one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .) and a EPO receptor activator (ESAs).
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • ESAs EPO receptor activator
  • one or more ActRII antagonist agents of the disclosure may be used in combination with ESAs to reduce the required dose of these activators in patients that are susceptible to adverse effects of ESAs. These methods may be used for therapeutic and prophylactic treatments of a patient.
  • One or more ActRII antagonist agents of the disclosure may be used in combination with EPO receptor activators to achieve an increase in red blood cells, particularly at lower dose ranges. This may be beneficial in reducing the known off-target effects and risks associated with high doses of EPO receptor activators.
  • the primary adverse effects of ESAs include, for example, an excessive increase in the hematocrit or hemoglobin levels and polycythemia. Elevated hematocrit levels can lead to hypertension (more particularly aggravation of hypertension) and vascular thrombosis.
  • an ActRII antagonist of the present disclosure may be beneficial for a patient in which administration of a normal to increased (>300 IU/kg/week) dose of ESA does not result in the increase of hemoglobin level up to the target level.
  • Patients with an inadequate ESA response are found for all types of anemia, but higher numbers of non-responders have been observed particularly frequently in patients with cancers and patients with end-stage renal disease.
  • An inadequate response to ESA can be either constitutive (observed upon the first treatment with ESA) or acquired (observed upon repeated treatment with ESA).
  • one or more ActRII antagonist agents of the disclosure may be used in combination with hepcidin, a hepcidin analog, or a hepcidin receptor activator for treating patients with SCD, particularly for complications associated with iron overload.
  • hepcidin is considered a master regulator of iron metabolism by virtue of its ability to induce the degradation of ferroportin, an iron-export protein localized on absorptive enterocytes, hepatocytes, and macrophages.
  • hepcidin reduces availability of extracellular iron, so hepcidin, hepcidin analogs, or hepcidin receptor activators may be beneficial in the treatment of patients with SCD, particularly for complications associated with iron overload.
  • the present disclosure provides methods for managing a patient that has been treated with, or is a candidate to be treated with, one or more one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .) by measuring one or more hematologic parameters in the patient.
  • the hematologic parameters may be used to evaluate appropriate dosing for a patient who is a candidate to be treated with the antagonist of the present disclosure, to monitor the hematologic parameters during treatment, to evaluate whether to adjust the dosage during treatment with one or more antagonist of the disclosure, and/or to evaluate an appropriate maintenance dose of one or more antagonists of the disclosure.
  • dosing with one or more ActRII antagonist agents of the disclosure may be reduced, delayed or terminated.
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • Hematologic parameters that may be measured in accordance with the methods provided herein include, for example, red blood cell levels, blood pressure, iron stores, and other agents found in bodily fluids that correlate with increased red blood cell levels, using art recognized methods. Such parameters may be determined using a blood sample from a patient. Increases in red blood cell levels, hemoglobin levels, and/or hematocrit levels may cause increases in blood pressure.
  • hematologic parameters are outside the normal range or on the high side of normal in a patient who is a candidate to be treated with one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .), then onset of administration of the one or more antagonists of the disclosure may be delayed until the hematologic parameters have returned to a normal or acceptable level either naturally or via therapeutic intervention. For example, if a candidate patient is hypertensive or pre-hypertensive, then the patient may be treated with a blood pressure lowering agent in order to reduce the patient's blood pressure.
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • Any blood pressure lowering agent appropriate for the individual patient's condition may be used including, for example, diuretics, adrenergic inhibitors (including alpha blockers and beta blockers), vasodilators, calcium channel blockers, angiotensin-converting enzyme (ACE) inhibitors, or angiotensin II receptor blockers.
  • Blood pressure may alternatively be treated using a diet and exercise regimen.
  • the patient may be treated with an appropriate regimen of diet and/or iron supplements until the patient's iron stores have returned to a normal or acceptable level.
  • administration of the one or more antagonists of the disclosure may be delayed until the levels have returned to a normal or acceptable level.
  • one or more hematologic parameters are outside the normal range or on the high side of normal in a patient who is a candidate to be treated with one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .), then the onset of administration may not be delayed.
  • the dosage amount or frequency of dosing of the one or more antagonists of the disclosure may be set at an amount that would reduce the risk of an unacceptable increase in the hematologic parameters arising upon administration of the one or more antagonists of the disclosure.
  • a therapeutic regimen may be developed for the patient that combines one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .) with a therapeutic agent that addresses the undesirable level of the hematologic parameter.
  • a therapeutic regimen may be designed involving administration of one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .) and a blood pressure lowering agent.
  • a therapeutic regimen may be developed involving one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .) and iron supplementation.
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • baseline parameter(s) for one or more hematologic parameters may be established for a patient who is a candidate to be treated with one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .) and an appropriate dosing regimen established for that patient based on the baseline value(s).
  • ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • established baseline parameters based on a patient's medical history could be used to inform an appropriate antagonist dosing regimen for a patient.
  • a patient's baseline values for one or more hematologic parameters prior to treatment with one or more ActRII antagonist agents of the disclosure may also be used as the relevant comparative values for monitoring any changes to the hematologic parameters during treatment with the one or more antagonists of the disclosure.
  • one or more hematologic parameters are measured in patients who are being treated with a one or more ActRII antagonist agents of the disclosure (e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .).
  • the hematologic parameters may be used to monitor the patient during treatment and permit adjustment or termination of the dosing with the one or more antagonists of the disclosure or additional dosing with another therapeutic agent.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • the dose of the one or more antagonists of the disclosure may be reduced in amount or frequency in order to decrease the effects of the one or more antagonists of the disclosure on the one or more hematologic parameters.
  • one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • the dosing of the one or more antagonists of the disclosure may be terminated either temporarily, until the hematologic parameter(s) return to an acceptable level, or permanently.
  • the dosing may be terminated if one or more hematologic parameters are not brought within an acceptable range after reducing the dose or frequency of administration of the one or more antagonists of the disclosure.
  • the patient may be dosed with an additional therapeutic agent that addresses the undesirable level in the hematologic parameter(s), such as, for example, a blood pressure lowering agent or an iron supplement.
  • an additional therapeutic agent that addresses the undesirable level in the hematologic parameter(s), such as, for example, a blood pressure lowering agent or an iron supplement.
  • a patient being treated with one or more ActRII antagonist agents of the disclosure e.g., a GDF-ActRII antagonist, an ActRIIA polypeptide, an ActRIIB polypeptide, a GDF trap, etc .
  • dosing with the one or more antagonists of the disclosure may continue at the same level and a blood-pressure-lowering agent is added to the treatment regimen, dosing with the one or more antagonist of the disclosure may be reduced ( e.g., in amount and/or frequency) and a blood-pressure-lowering agent is added to the treatment regimen, or dosing with the one or more antagonist of the disclosure may be terminated and the patient may be treated with a blood-pressure-lowering agent.
  • one or more ActRII antagonist agents of the disclosure can be administered alone or as a component of a pharmaceutical formulation (also referred to as a therapeutic composition or pharmaceutical composition).
  • a pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient (e.g., an agent of the present disclosure) contained therein to be effective and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • the subject compounds may be formulated for administration in any convenient way for use in human or veterinary medicine.
  • one or more agents of the present disclosure may be formulated with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is generally nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, and/or preservative.
  • pharmaceutical formulations for use in the present disclosure are in a pyrogen-free, physiologically-acceptable form when administered to a subject.
  • Therapeutically useful agents other than those described herein, which may optionally be included in the formulation as described above, may be administered in combination with the subject agents in the methods of the present disclosure.
  • compositions suitable for parenteral administration may comprise one or more agents of the disclosure in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use.
  • Injectable solutions or dispersions may contain antioxidants, buffers, bacteriostats, suspending agents, thickening agents, or solutes which render the formulation isotonic with the blood of the intended recipient.
  • suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical formulations of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc .), vegetable oils (e.g., olive oil), injectable organic esters ( e.g., ethyl oleate), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • a therapeutic method of the present disclosure includes administering the pharmaceutical composition systemically, or locally, from an implant or device. Further, the pharmaceutical composition may be encapsulated or injected in a form for delivery to a target tissue site (e.g., bone marrow or muscle).
  • compositions of the present disclosure may include a matrix capable of delivering one or more of the agents of the present disclosure to a target tissue site (e.g., bone marrow or muscle), providing a structure for the developing tissue and optimally capable of being resorbed into the body. For example, the matrix may provide slow release of one or more agents of the present disclosure.
  • Such matrices may be formed of materials presently in use for other implanted medical applications.
  • the choice of matrix material may be based on one or more of: biocompatibility, biodegradability, mechanical properties, cosmetic appearance, and interface properties.
  • Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, and polyanhydrides.
  • Other potential materials are biodegradable and biologically well-defined, including, for example, bone or dermal collagen.
  • Further matrices are comprised of pure proteins or extracellular matrix components.
  • Other potential matrices are non-biodegradable and chemically defined, including, for example, sintered hydroxyapatite, bioglass, aluminates, or other ceramics.
  • Matrices may be comprised of combinations of any of the above mentioned types of material including, for example, polylactic acid and hydroxyapatite or collagen and tricalciumphosphate.
  • the bioceramics may be altered in composition (e.g., calcium-aluminate-phosphate) and processing to alter one or more of pore size, particle size, particle shape, and biodegradability.
  • compositions of the present disclosure can be administered orally, for example, in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis such as sucrose and acacia or tragacanth), powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, or an elixir or syrup, or pastille (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and/or a mouth wash, each containing a predetermined amount of a compound of the present disclosure and optionally one or more other active ingredients.
  • a compound of the present disclosure and optionally one or more other active ingredients may also be administered as a bolus, electuary, or paste.
  • one or more compounds of the present disclosure may be mixed with one or more pharmaceutically acceptable carriers including, for example, sodium citrate, dicalcium phosphate, a filler or extender (e.g., a starch, lactose, sucrose, glucose, mannitol, and silicic acid), a binder (e.g.
  • carboxymethylcellulose an alginate, gelatin, polyvinyl pyrrolidone, sucrose, and acacia
  • a humectant e.g., glycerol
  • a disintegrating agent e.g., agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, a silicate, and sodium carbonate
  • a solution retarding agent e.g. paraffin
  • an absorption accelerator e.g.
  • the pharmaceutical formulation may also comprise a buffering agent.
  • a wetting agent e.g., cetyl alcohol and glycerol monostearate
  • an absorbent e.g., kaolin and bentonite clay
  • a lubricant e.g., a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
  • a coloring agent e.g., a quaternary ammonium compound
  • a wetting agent e.g., cetyl alcohol and glycerol monostearate
  • an absorbent e.g., kaolin and bentonite clay
  • a lubricant e.g., a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
  • a coloring agent e.g., a coloring agent, and mixtures thereof.
  • the pharmaceutical formulation may also comprise
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using one or more excipients including, e.g., lactose or a milk sugar as well as a high molecular-weight polyethylene glycol.
  • Liquid dosage forms for oral administration of the pharmaceutical composition may include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage form may contain an inert diluent commonly used in the art including, for example, water or other solvent, a solubilizing agent and/or emulsifier [ e.g., ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, or 1,3-butylene glycol, an oil ( e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oil), glycerol, tetrahydrofuryl alcohol, a polyethylene glycol, a fatty acid ester of sorbitan, and mixtures thereof].
  • an inert diluent commonly used in the art including, for example, water
  • the oral formulation can also include an adjuvant including, for example, a wetting agent, an emulsifying and suspending agent, a sweetening agent, a flavoring agent, a coloring agent, a perfuming agent, a preservative agent, and combinations thereof.
  • an adjuvant including, for example, a wetting agent, an emulsifying and suspending agent, a sweetening agent, a flavoring agent, a coloring agent, a perfuming agent, a preservative agent, and combinations thereof.
  • Suspensions in addition to the active compounds, may contain suspending agents including, for example, an ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and combinations thereof.
  • suspending agents including, for example, an ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, a sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and combinations thereof.
  • antibacterial and antifungal agents including, for example, paraben, chlorobutanol, and phenol sorbic acid.
  • an isotonic agent including, for example, a sugar or sodium chloride into the compositions.
  • prolonged absorption of an injectable pharmaceutical form may be brought about by the inclusion of an agent that delays absorption, including, for example, aluminum monostearate and gelatin.
  • the dosage regimen will be determined by the attending physician considering various factors which modify the action of the one or more of the agents of the present disclosure.
  • the various factors include, but are not limited to, the patient's red blood cell count, hemoglobin level, the desired target red blood cell count, the patient's age, the patient's sex, the patient's diet, the severity of any disease that may be contributing to a depressed red blood cell level, the time of administration, and other clinical factors.
  • the addition of other known active agents to the final composition may also affect the dosage. Progress can be monitored by periodic assessment of one or more of red blood cell levels, hemoglobin levels, reticulocyte levels, and other indicators of the hematopoietic process.
  • the present disclosure also provides gene therapy for the in vivo production of one or more of the agents of the present disclosure.
  • Such therapy would achieve its therapeutic effect by introduction of the agent sequences into cells or tissues having one or more of the disorders as listed above.
  • Delivery of the agent sequences can be achieved, for example, by using a recombinant expression vector such as a chimeric virus or a colloidal dispersion system.
  • Preferred therapeutic delivery of one or more of agent sequences of the disclosure is the use of targeted liposomes.
  • RNA virus e.g., a retrovirus
  • the retroviral vector may be a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous sarcoma virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous sarcoma virus
  • a number of additional retroviral vectors can incorporate multiple genes.
  • Retroviral vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target-specific by attaching, for example, a sugar, a glycolipid, or a protein. Preferred targeting is accomplished by using an antibody.
  • specific polynucleotide sequences can be inserted into the retroviral genome or attached to a viral envelope to allow target specific delivery of the retroviral vector containing one or more of the agents of the present disclosure.
  • tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes (gag, pol, and env), by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion system Another targeted delivery system for one or more of the agents of the present disclosure is a colloidal dispersion system.
  • Colloidal dispersion systems include, for example, macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this disclosure is a liposome.
  • Liposomes are artificial membrane vesicles which are useful as delivery vehicles in vitro and in vivo. RNA, DNA, and intact virions can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form [see, e.g., Fraley, et al. (1981) Trends Biochem. Sci., 6:77 ].
  • Methods for efficient gene transfer using a liposome vehicle are known in the art [see, e.g., Mannino, et al. (1988) Biotechniques, 6:
  • the composition of the liposome is usually a combination of phospholipids, which may include a steroid (e.g. cholesterol).
  • a steroid e.g. cholesterol
  • the physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.
  • Other phospholipids or other lipids may also be used, including, for example a phosphatidyl compound (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipid, cerebroside, or a ganglioside), egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine.
  • the targeting of liposomes is also possible based on, for example, organ specificity, cell specificity, and organelle specificity and is known in the art.
  • Applicants constructed a soluble ActRIIA fusion protein that has the extracellular domain of human ActRIIa fused to a human or mouse Fc domain with a minimal linker in between.
  • the constructs are referred to as ActRIIA-hFc and ActRIIA-mFc, respectively.
  • ActRIIA-hFc is shown below as purified from CHO cell lines (SEQ ID NO:22):
  • the ActRIIA-hFc and ActRIIA-mFc proteins were expressed in CHO cell lines.
  • the selected form employs the TPA leader and has the following unprocessed amino acid sequence:
  • This polypeptide is encoded by the following nucleic acid sequence:
  • ActRIIA-hFc and ActRIIA-mFc showed a high affinity for ligands, particularly activin A.
  • GDF-11 or activin A were immobilized on a BiacoreTM CM5 chip using standard amine-coupling procedure.
  • ActRIIA-hFc and ActRIIA-mFc proteins were loaded onto the system, and binding was measured.
  • ActRIIA-hFc bound to activin with a dissociation constant (K D ) of 5 x 10 -12 and bound to GDF11 with a K D of 9.96 x 10 -9 . See Figure 4 .
  • ActRIIA-mFc behaved similarly.
  • the ActRIIA-hFc was very stable in pharmacokinetic studies. Rats were dosed with 1 mg/kg, 3 mg/kg, or 10 mg/kg of ActRIIA-hFc protein, and plasma levels of the protein were measured at 24, 48, 72, 144 and 168 hours. In a separate study, rats were dosed at 1 mg/kg, 10 mg/kg, or 30 mg/kg.
  • ActRIIA-hFc had an 11-14 day serum half-life, and circulating levels of the drug were quite high after two weeks (11 ⁇ g/ml, 110 ⁇ g/ml, or 304 ⁇ g/ml for initial administrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively.)
  • the plasma half-life was substantially greater than 14 days, and circulating levels of the drug were 25 ⁇ g/ml, 304 ⁇ g/ml, or 1440 ⁇ g/ml for initial administrations of 1 mg/kg, 10 mg/kg, or 30 mg/kg, respectively.
  • ActRIIA-hFc fusion protein was expressed in stably transfected CHO-DUKX B11 cells from a pAID4 vector (SV40 ori/enhancer, CMV promoter), using a tissue plasminogen leader sequence of SEQ ID NO:9.
  • the protein purified as described above in Example 1, had a sequence of SEQ ID NO:22.
  • the Fc portion is a human IgG1 Fc sequence, as shown in SEQ ID NO:22. Protein analysis reveals that the ActRIIA-hFc fusion protein is formed as a homodimer with disulfide bonding.
  • the CHO-cell-expressed material has a higher affinity for activin B ligand than that reported for an ActRIIa-hFc fusion protein expressed in human 293 cells [see, del Re et al. (2004) J Biol Chem. 279(51):53126-53135 ]. Additionally, the use of the TPA leader sequence provided greater production than other leader sequences and, unlike ActRIIA-Fc expressed with a native leader, provided a highly pure N-terminal sequence. Use of the native leader sequence resulted in two major species of ActRIIA-Fc, each having a different N-terminal sequence.
  • the study employed four groups of five male and five female cynomolgus monkeys each, with three per sex per group scheduled for termination on Day 29, and two per sex per group scheduled for termination on Day 57.
  • Each animal was administered the vehicle (Group 1) or ActRIIA-Fc at doses of 1, 10, or 30 mg/kg (Groups 2, 3 and 4, respectively) via intravenous (IV) injection on Days 1, 8, 15, and 22.
  • the dose volume was maintained at 3 mL/kg.
  • Various measures of red blood cell levels were assessed two days prior to the first administration and at days 15, 29, and 57 (for the remaining two animals) after the first administration.
  • the ActRIIA-hFc caused statistically significant increases in mean red blood cell parameters [red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT)] for males and females, at all dose levels and time points throughout the study, with accompanying elevations in absolute and relative reticulocyte counts (ARTC; RTC). See Figures 5-8 .
  • red blood cell counts and hemoglobin levels are roughly equivalent in magnitude to effects reported with erythropoietin.
  • the onset of these effects is more rapid with ActRIIA-Fc than with erythropoietin.
  • the ActRIIA-hFc fusion protein described in Example 1 was administered to human subjects in a randomized, double-blind, placebo-controlled study that was conducted to evaluate, primarily, the safety of the protein in healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ActRIIA-hFc or placebo (5 active: 1 placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously (IV) and 0.03 to 0.1 mg/kg subcutaneously (SC). All subjects were followed for 120 days. In addition to pharmacokinetic (PK) analyses, the biologic activity of ActRIIA-hFc was also assessed by measurement of biochemical markers of bone formation and resorption as well as FSH levels.
  • PK pharmacokinetic
  • hemoglobin and RBC numbers were examined in detail for all subjects over the course of the study and compared to the baseline levels. Platelet counts were compared over the same time as the control. There were no clinically significant changes from the baseline values over time for the platelet counts.
  • PK Pharmacokinetic analysis of ActRIIA-hFc revealed a linear profile with dose, and a mean half-life of approximately 25-32 days.
  • AUC area-under-curve
  • ActRIIA-hFc caused a rapid, sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase (BAP), which is a marker for anabolic bone growth, and a dose-dependent decrease in C-terminal type 1 collagen telopeptide and tartrate-resistant acid phosphatase 5b levels, which are markers for bone resorption.
  • BAP bone-specific alkaline phosphatase
  • Other markers such as P1NP showed inconclusive results.
  • BAP levels showed near-saturating effects at the highest dosage of drug, indicating that half-maximal effects on this anabolic bone biomarker could be achieved at a dosage of 0.3 mg/kg, with increases ranging up to 3 mg/kg.
  • the EC50 was 51,465 (day*ng/ml) (see Figure 11 ). These bone biomarker changes were sustained for approximately 120 days at the highest dose levels tested. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin.
  • ActRIIA-hFc showed a dose-dependent effect on red blood cell counts and reticulocyte counts.
  • Example 5 Treatment of an Anemic Patient with ActRIIA-hFc
  • a clinical study was designed to treat patients with multiple doses of ActRIIA-hFc, at three dose levels of 0.1 mg/kg, 0.3 mg/kg, and 1.0 mg/kg, with dosing to occur every 30 days.
  • Normal healthy subjects in the trial exhibited an increase in hemoglobin and hematocrit that is consistent with the increases seen in the Phase I clinical trial reported in Example 4, except that in some instances the hemoglobin (Hg) and hematocrit (Hct) are elevated beyond the normal range.
  • An anemic patient with hemoglobin levels of approximately 7.5 g/dL also received two doses at the 1 mg/kg level, resulting in a hemoglobin level of approximately 10.5 g/dL after two months.
  • the patient's anemia was a microcytic anemia, thought to be caused by chronic iron deficiency.
  • ActRIIA-Fc fusion proteins have been further demonstrated to be effective in increasing red blood cell levels in various models of anemia including, for example, chemotherapy-induced anemia and anemia associated with chronic kidney disease (see, e.g., U.S. Patent Application Publication No. 2010/0028331 ).
  • ActRIIA variants that may be used according to the methods described herein are described in the International Patent Application published as WO2006/012627 (see e.g., pp. 55-58), incorporated herein by reference in its entirety.
  • An alternative construct may have a deletion of the C-terminal tail (the final 15 amino acids of the extracellular domain of ActRIIA. The sequence for such a construct is presented below (Fc portion underlined) (SEQ ID NO:28):
  • the constructs are referred to as ActRIIB-hFc and ActRIIB-mFc, respectively.
  • the ActRIIB-hFc and ActRIIB-mFc proteins were expressed in CHO cell lines. Three different leader sequences were considered:
  • the selected form employs the TPA leader and has the following unprocessed amino acid sequence (SEQ ID NO: 31):
  • This polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO:32):
  • N-terminal sequencing of the CHO-cell-produced material revealed a major sequence of -GRGEAE (SEQ ID NO:33). Notably, other constructs reported in the literature begin with an -SGR... sequence.
  • Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • ActRIIB-Fc fusion proteins were also expressed in HEK293 cells and COS cells. Although material from all cell lines and reasonable culture conditions provided protein with muscle-building activity in vivo, variability in potency was observed perhaps relating to cell line selection and/or culture conditions.
  • the background ActRIIB-Fc fusion has the sequence of SEQ ID NO:29.
  • HEK293T cells were produced in HEK293T cells by transient transfection.
  • HEK293T cells were set up at 6x10 5 cells/ml in Freestyle (Invitrogen) media in 250ml volume and grown overnight. Next day, these cells were treated with DNA:PEI (1:1) complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml media was added and cells were grown for 7 days. Conditioned media was harvested by spinning down the cells and concentrated.
  • Mutants were purified using a variety of techniques, including, for example, a protein A column, and eluted with low pH (3.0) glycine buffer. After neutralization, these were dialyzed against PBS.
  • Mutants were also produced in CHO cells by similar methodology. Mutants were tested in binding assays and/or bioassays described in WO 2008/097541 and WO 2006/012627 incorporated by reference herein. In some instances, assays were performed with conditioned medium rather than purified proteins. Additional variations of ActRIIB are described in U.S. Patent No. 7,842,663 .
  • Example 8 ActRIIB-Fc Stimulates Erythropoiesis in Non-Human Primates
  • Cynomolgus monkeys were allocated into seven groups (6/sex/group) and administered ActRIIB(20-134)-hFc as a subcutaneous injection at dosages of 0.6, 3, or 15 mg/kg every 2 weeks or every 4 weeks over a 9-month period.
  • the control group (6/sex/group) received the vehicle at the same dose volume (0.5 ml/kg/dose) as ActRIIB(20-134)-hFc-treated animals. Animals were monitored for changes in general clinical pathology parameters (e.g., hematology, clinical chemistry, coagulation, and urinalysis).
  • Hematology, coagulation, and clinical chemistry parameters were evaluated twice prior to initiation of dosing and on Days 59, 143, 199, 227, and on Days 267 (for groups dosed every 4 weeks) or 281 (for groups dosed every 2 weeks). The evaluations on Days 267/281 occurred 2 weeks after the final dose was administered.
  • ActRIIB(20-134)-hFc resulted in non-adverse, dose-related changes to hematology parameters in male and female monkeys. These changes included increased red blood cell count, reticulocyte count and red cell distribution width and decreased mean corpuscular volume, mean corpuscular hemoglobin, and platelet count.
  • RBC count was increased at all dose levels, and the magnitude of increase was generally comparable whether ActRIIB(20-134)-hFc was administered every 2 weeks or every 4 weeks.
  • Mean RBC count was increased at all time points between Days 59 and 267/281 (except RBC count was not increased in group 2 males [0.6 mg/kg every 2 weeks] on Day 281).
  • RBC count was increased at ⁇ 3 mg/kg every 2 weeks and the changes occurred between Days 143 and 281; at 15 mg/kg every 4 weeks mean RBC count was increased between Days 59 and 267.
  • a binding relative to GDF11 and/or myostatin (as a consequence of a leucine-to-aspartate substitution at position 79 in SEQ ID NO:1) was fused to a human or mouse Fc domain with a minimal linker (three glycine amino acids) in between.
  • the constructs are referred to as ActRIIB(L79D 20-134)-hFc and ActRIIB(L79D 20-134)-mFc, respectively.
  • the GDF trap ActRIIB(L79D 20-134)-hFc is shown below as purified from CHO cell lines (SEQ ID NO:36).
  • the ActRIIB-derived portion of the GDF trap has an amino acid sequence set forth below (SEQ ID NO: 37), and that portion could be used as a monomer or as a non-Fc fusion protein as a monomer, dimer, or greater-order complex.
  • the GDF trap protein was expressed in CHO cell lines. Three different leader sequences were considered:
  • the selected form employs the TPA leader and has the following unprocessed amino acid sequence:
  • This polypeptide is encoded by the following nucleic acid sequence (SEQ ID NO:39):
  • Purification could be achieved by a series of column chromatography steps, including, for example, three or more of the following, in any order: protein A chromatography, Q sepharose chromatography, phenylsepharose chromatography, size exclusion chromatography, and cation exchange chromatography.
  • the purification could be completed with viral filtration and buffer exchange.
  • the cell culture medium is passed over a protein A column, washed in 150 mM Tris/NaCl (pH 8.0), then washed in 50 mM Tris/NaCl (pH 8.0) and eluted with 0.1 M glycine, pH 3.0.
  • the low pH eluate is kept at room temperature for 30 minutes as a viral clearance step.
  • the eluate is then neutralized and passed over a Q-sepharose ion-exchange column and washed in 50 mM Tris pH 8.0, 50 mM NaCl, and eluted in 50 mM Tris pH 8.0, with an NaCl concentration between 150 mM and 300 mM.
  • the eluate is then changed into 50 mM Tris pH 8.0, 1.1 M ammonium sulfate and passed over a phenyl sepharose column, washed, and eluted in 50 mM Tris pH 8.0 with ammonium sulfate between 150 and 300 mM.
  • the eluate is dialyzed and filtered for use.
  • A-204 reporter gene assay was used to evaluate the effects of ActRIIB-Fc proteins and GDF traps on signaling by GDF-11 and activin A.
  • Cell line human rhabdomyosarcoma (derived from muscle).
  • Reporter vector pGL3(CAGA)12 (described in Dennler et al, 1998, EMBO 17: 3091-3100 ).
  • the CAGA12 motif is present in TGF-beta responsive genes (e.g., PAI-1 gene), so this vector is of general use for factors signaling through SMAD2 and 3.
  • activin A showed 10-fold stimulation of reporter gene expression and an ED50 ⁇ 2 ng/ml.
  • GDF-11 16 fold stimulation
  • ED50 ⁇ 1.5 ng/ml.
  • ActRIIB(20-134) is a potent inhibitor of activin A, GDF-8, and GDF-11 activity in this assay. As described below, ActRIIB variants were also tested in this assay.
  • ActRIIB-Fc proteins and GDF traps were tested in a cell-based assay as described above. Results are summarized in the table below. Some variants were tested in different C-terminal truncation constructs. As discussed above, truncations of five or fifteen amino acids caused reduction in activity. The GDF traps (L79D and L79E variants) showed substantial loss of activin A inhibition while retaining almost wild-type inhibition of GDF-11. Soluble ActRIIB-Fc binding to GDF11 and Activin A: ActRIIB-Fc Variations Portion of ActRIIB (corresponds to amino acids of SEQ ID NO:1) GDF11 Inhibition Activity Activin Inhibition Activity R64 20-134 +++ (approx.
  • ActRIIB(20-134)-Fc has a serum half-life of approximately 70 hours.
  • ActRIIB(A24N 20-134)-Fc has a serum half-life of approximately 100-150 hours.
  • the A24N variant has activity in the cell-based assay (above) and in vivo assays (below) that is equivalent to the wild-type molecule. Coupled with the longer half-life, this means that over time an A24N variant will give greater effect per unit of protein than the wild-type molecule.
  • the A24N variant, and any of the other variants tested above, may be combined with the GDF trap molecules, such as the L79D or L79E variants.
  • Example 12 GDF-11 and Activin A Binding.
  • the ActRIIB-Fc variants or wild-type protein were captured onto the system using an anti-hFc antibody. Ligands were injected and flowed over the captured receptor proteins. Results are summarized in the tables below. Ligand-binding specificity IIB variants.
  • Example 13 A GDF Trap Increases Red Blood Cell Levels in vivo
  • GDF trap ActRIIB polypeptide
  • Treatment with the GDF trap did not have a statistically significant effect on the number of white blood cells (WBC) compared to the vehicle controls.
  • Red blood cell (RBC) numbers were increased in the treated group relative to the controls (see table below). Both the hemoglobin content (HGB) and hematocrit (HCT) were also increased due to the additional red blood cells.
  • the average width of the red blood cells (RDWc) was higher in the treated animals, indicating an increase in the pool of immature red blood cells. Therefore, treatment with the GDF trap leads to increases in red blood cells, with no distinguishable effects on white blood cell populations.
  • Example 14 A GDF Trap is Superior to ActRIIB-Fc for Increasing Red Blood Cell Levels in vivo.
  • mice Nineteen-week-old male C57BL/6NTac mice were randomly assigned to one of three groups. Mice were dosed with vehicle (10 mM Tris-buffered saline, TBS), wild-type ActRIIB(20-134)-mFc, or GDF trap ActRIIB(L79D 20-134)-hFc by subcutaneous injection twice per week for three weeks. Blood was collected by cheek bleed at baseline and after three weeks of dosing and analyzed for cell distribution using a hematology analyzer (HM2, Abaxis, Inc.)
  • HM2 hematology analyzer
  • a GDF trap referred to as ActRIIB(L79D 20-134)-hFc was generated by N-terminal fusion of TPA leader to the ActRIIB extracellular domain (residues 20-134 in SEQ ID NO:1) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO:1) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) ( Figure 16 ).
  • a nucleotide sequence corresponding to this fusion protein is shown in Figure 17 .
  • a GDF trap with truncated ActRIIB extracellular domain referred to as ActRIIB(L79D 25-131)-hFc, was generated by N-terminal fusion of TPA leader to truncated extracellular domain (residues 25-131 in SEQ ID NO:1) containing a leucine-to-aspartate substitution (at residue 79 in SEQ ID NO:1) and C-terminal fusion of human Fc domain with minimal linker (three glycine residues) ( Figure 18 ).
  • a nucleotide sequence corresponding to this fusion protein is shown in Figure 19 .
  • Example 18 GDF Trap with a Truncated ActRIIB Extracellular Domain Increases Proliferation of Erythroid Progenitors in Mice
  • ActRIIB(L79D 25-131)-hFc was evaluated to determine its effect on proliferation of erythroid progenitors.
  • CFU-E colony-forming unit-erythroid
  • BFU-E burst forming unit-erythroid
  • Methylcellulose-based medium for BFU-E determination included recombinant murine stem cell factor, interleukin-3, and interleukin-6, which were not present in methylcellulose medium for CFU-E determination (MethoCult M3334, Stem Cell Technologies), while both media contained erythropoietin, among other constituents.
  • MethodhoCult M3334, Stem Cell Technologies included recombinant murine stem cell factor, interleukin-3, and interleukin-6, which were not present in methylcellulose medium for CFU-E determination
  • both media contained erythropoietin, among other constituents.
  • the number of colonies were determined in duplicate culture plates derived from each tissue sample, and statistical analysis of the results was based on the number of mice per treatment group.
  • GDF trap fusion proteins have been further demonstrated to be effective in increasing red blood cell levels in various models of anemia including, for example, chemotherapy-induced anemia, nephrectomy-induced anemia, and in a blood loss anemia (see, e.g., International Patent Application Publication No. WO 2010/019261 ).
  • Vehicle-treated monkeys exhibited decreased levels of red blood cells, hematocrit, and hemoglobin at all post-treatment time points, an expected effect of repeated blood sampling.
  • treatment with ActRIIB(L79D 20-134)-hFc or ActRIIB(L79D 25-131)-hFc increased these parameters by the first post-treatment time point (Day 3) and maintained them at substantially elevated levels for the duration of the study ( Figures 24-26 ).
  • reticulocyte levels in monkeys treated with ActRIIB(L79D 20-134)-hFc or ActRIIB(L79D 25-131)-hFc were substantially increased at Days 8, 15, and 29 compared to vehicle ( Figure 27 ). This result demonstrates that GDF trap treatment increased production of red blood cell precursors, resulting in elevated red blood cell levels.
  • ActRIIB5 an alternate, soluble form of ActRIIB (designated ActRIIB5), in which exon 4, including the ActRIIB transmembrane domain, has been replaced by a different C-terminal sequence (see, e.g., WO 2007/053775 ).
  • An leucine-to-aspartate substitution, or other acidic substitutions, may be performed at native position 79 (underlined) as described to construct the variant ActRIIB5(L79D), which has the following sequence:
  • This variant may be connected to human Fc ( double underline ) with a TGGG linker ( single underline ) to generate a human ActRIIB5(L79D)-hFc fusion protein with the following sequence:
  • This construct may be expressed in CHO cells.
  • EPO induces formation of red blood cells by increasing the proliferation of erythroid precursors, whereas GDF traps could potentially affect formation of red blood cells in ways that complement or enhance EPO's effects. Therefore, Applicants investigated the effect of combined treatment with EPO and ActRIIB(L79D 25-131)-hFc on erythropoietic parameters.
  • Male C57BL/6 mice (9 weeks old) were given a single i.p. injection of recombinant human EPO alone (epoetin alfa, 1800 units/kg), ActRIIB(L79D 25-131)-hFc alone (10 mg/kg), both EPO and ActRIIB(L79D 25-131)-hFc, or vehicle (Tris-buffered saline). Mice were euthanized 72 h after dosing for collection of blood, spleens, and femurs.
  • Spleens and femurs were processed to obtain erythroid precursor cells for flow cytometric analysis. After removal, the spleen was minced in Iscove's modified Dulbecco's medium containing 5% fetal bovine serum and mechanically dissociated by pushing through a 70- ⁇ m cell strainer with the plunger from a sterile 1-mL syringe. Femurs were cleaned of any residual muscle or connective tissue and ends were trimmed to permit collection of marrow by flushing the remaining shaft with Iscove's modified Dulbecco's medium containing 5% fetal bovine serum through a 21-gauge needle connected to a 3-mL syringe.
  • Cell suspensions were centrifuged (2000 rpm for 10 min) and the cell pellets resuspended in PBS containing 5% fetal bovine serum.
  • Cells (10 6 ) from each tissue were incubated with anti-mouse IgG to block nonspecific binding, then incubated with fluorescently labeled antibodies against mouse cell-surface markers CD71 (transferrin receptor) and Ter119 (an antigen associated with cell-surface glycophorin A), washed, and analyzed by flow cytrometry. Dead cells in the samples were excluded from analysis by counterstaining with propidium iodide.
  • Erythroid differentiation in spleen or bone marrow was assessed by the degree of CD71 labeling, which decreases over the course of differentiation, and Ter119 labeling, which is increased during terminal erythroid differentiation beginning with the proerythroblast stage ( Socolovsky et al., 2001, Blood 98:3261-3273 ; Ying et al., 2006, Blood 108:123-133 ).
  • flow cytometry was used to determine the number of proerythroblasts (CD71 hight Ter119 low ), basophilic erythroblasts (CD71 high Ter11 high ), polychromatophilic + orthochromatophilic erythroblasts (CD71 med Ter119 high ), and late orthochromatophilic erythroblasts + reticulocytes (CD71 low Ter119 high ), as described.
  • a GDF trap with a truncated ActRIIB extracellular domain can be administered in combination with EPO to synergistically increase red blood cell formation in vivo.
  • a GDF trap can moderate the strong proliferative effect of an EPO receptor activator alone and still permit target levels of red blood cells to be attained with lower doses of an EPO receptor activator, thereby avoiding potential adverse effects or other problems associated with higher levels of EPO receptor activation.
  • SCD sickle-cell disease
  • mice homozygous for the human ⁇ S allele exhibit the major features (e.g., severe hemolytic anemia, irreversibly sickled red cells, vascular (vaso) occlusion, and multi-organ pathology) found in humans with sickle-cell disease [see, e.g., Wu et al., (2006) Blood, 108(4): 1183-1188 ; Ryan et al. (1997) Science 278: 873-876 ].
  • major features e.g., severe hemolytic anemia, irreversibly sickled red cells, vascular (vaso) occlusion, and multi-organ pathology
  • SCD mice ( ⁇ S / ⁇ S ) at 3 months of age were randomly assigned to receive ActRIIB(L79D 25-131)-mFc (1 mg/kg or 10 mg/kg) or vehicle [Tris-buffered saline (TBS)] by subcutaneous injections twice weekly.
  • ActRIIB Li/kg or 10 mg/kg
  • TBS Tris-buffered saline
  • Non-symptomatic compound heterozygote ⁇ / ⁇ S
  • SCD mice had reduced RBC levels (-28%, P ⁇ 0.01) and hemoglobin levels (-14.5%, P ⁇ 0.05) and increased reticulocyte levels (+50%, P ⁇ 0.001) compared to the compound heterozygote mice, demonstrating that the SCD mice were severely anemic.
  • PS phosphatidylserine
  • ActRIIB(L79D 25-131)-mFc may be useful in the treatment of other complications associated with sickle-cell disease including, for example, splenic sequestration of red blood cells, which can result in splenic sequestration crisis and/or spenomegaly.
  • O 2 Hb oxyhemoglobin
  • COHb carboxyhemoglobin
  • a GDF trap comprising an ActRIIB extracellular domain can provide various therapeutic benefits in a murine model of SCD.
  • GDF trap therapy improved RBC morphology (i.e., reduced numbers of sickle shaped cells), reduced enlargement of the spleen, and reduced end organ damage in SCD patients.
  • ActRII antagonists e.g., GDF trap polypeptides
  • non-anemia complications of sickle-cell disease e.g., complications arising from vaso-occlusion
  • ActRII antagonists e.g., GDF trap polypeptides
  • GDF trap polypeptides can raise RBC levels by promoting use of endogenous iron stores via erythropoiesis and thus avoid iron overloading and its negative consequences.
  • ACS Acute chest syndrome
  • ActRIIB(L79D 25-131)-mFc pre-treatment significantly extended survival time (3-fold) SCD mice compared to vehicle pre-treatment.
  • ActRII antagonists e.g., GDF trap polypeptides
  • GDF trap polypeptides can be used to reduce acute intravascular hemolysis and provide increased resistance to naturally occurring ACS episodes in SCD patients.
  • SCD mice ( ⁇ S / ⁇ S ) were placed into four treatment groups: i) treatment with ActRIIB(L79D 25-131)-mFc (10 mg/kg, twice weekly, s.c.); ii) treatment with ActRIIB(L79D 25-131)-mFc (10 mg/kg, twice weekly, s.c.) and HU (100 mg/kg, twice weekly, i.p.); iii) treatment with HU (100 mg/kg, twice weekly, i.p.); and iv) TBS vehicle (control) for three months.
  • Non-symptomatic compound heterozygote ( ⁇ / ⁇ S ) litermates were treated similarly and used as controls to confirm disease in SCD ( ⁇ S / ⁇ S ) mice.
  • SCD mice had reduced RBC levels (-28%, P ⁇ 0.01 ) and hemoglobin levels (-14.5%, P ⁇ 0.05 ) and increased reticulocytes levels (+50%, P ⁇ 0.001 ) compared to compound heterozygote mice, indicating hemolysis characteristic of sickle-cell disease.
  • subjects were assessed for changes in various parameters.
  • ActRIIB(L79D 25-131)-mFc (alone) treatment resulted in significant increases in red blood cell levels (+20%, p ⁇ 0.01) and decreases in reticulocytes (-30%, p ⁇ 0.05) compared to vehicle-treated SCD mice.
  • Data from the combined ActRIIB(L79D 25-131)-mFc and HU treatment demonstrated additive beneficial effects in SCD mice compared to monotherapy.
  • the combination of ActRIIB(L79D 25-131)-mFc and HU resulted in a greater reduction in annexin V/PS exposure on peripheral blood cells than treatment with HU alone [-35.6% (p ⁇ 0.001) vs.
  • mice receiving the combination therapy (-55.0%, p ⁇ 0.01) compared to monotherapy with either HU (-48.4%, p ⁇ 0.05) or ActRIIB(L79D 25-131)-mFc (-40.8%, p ⁇ 0.05), all relative to vehicle-treated SCD mice.
  • Histological analysis further demonstrated a greater reduction in vascular congestion and damage in spleens and kidneys of mice receiving the ActRIIB(L79D 25-131)-mFc and HU combination therapy compared to mice receiving vehicle or HU monotherapy. See Figure 36 .
  • the histology demonstrated a greater reduction in alveolar thickening within the lungs of mice receiving the ActRIIB(L79D 25-131)-mFc and HU combination therapy compared to mice receiving the vehicle or HU monotherapy. See black arrows in Figure 36 .
  • the present disclosure includes the following aspects:

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